Glycoprotein VI antibodies and methods thereof

ABSTRACT

The present invention describes antibodies generated against platelet membrane glycoprotein VI-(GPVI), methods of producing the anti-GPVI antibodies, and the use of these antibodies as research and immunotherapeutic agents, in particular, as antithrombotic therapeutic agents.

This application claims the benefit of priority to U.S. ProvisionalApplication No. 60/566,171, filed Apr. 29, 2004, which is incorporatedherein by reference.

DESCRIPTION OF THE INVENTION

1. Field of the Invention

The present invention relates to antibodies generated against plateletmembrane glycoprotein VI (GPVI), fragments, or naturally-occurringvariants thereof, to methods of producing the anti-GPVI antibodies, andto the use of these antibodies as research and immunotherapuetic agents,in particular, as therapeutic agents for treating thrombosis and othervascular diseases.

2. Background of the Invention

Platelets are small, a nuclear blood cells that are essential tohemostatic control and wound healing. Circulating platelets are fairlyquiescent under normal conditions. However, when a blood vessel is tornor damaged, platelets are exposed to various factors that instigatecomplicated and interconnected cellular programs leading to bloodcoagulation and clot formation, which are reviewed in Mechanisms ofPlatelet Activation and Control, K. S. Authi, S. P. Watson, and V. V.Kakar (eds.) Plenum Press, 1993. The activation of these cellularprograms result in dramatic increases in membrane adhesive properties,platelet aggregation, and the release of vasoconstrictive andfibrinolytic factors. As a consequence, a clot forms at the site oftrauma, plugging any breach in the vessel wall and providing a substratefor fibroblast invasion and repair.

The early events in the clotting process can be functionally separatedinto two primary components: adhesion and activation. Adhesion is theprocess of “sticking” platelets to the injured vascular wall, whereasactivation initiates complex physiological changes inside the cell.Together, these two processes result in platelet aggregation, plugformation, and ultimately, in a mature clot. Although these events arecrucial in limiting blood loss to the site of injury, platelet adhesionand activation may also contribute to exacerbation of a diseased state.For example, clotting may cause blockage of diseased blood vessels,leading to ischemia and resulting in damage to vital tissues such as theheart and brain. The dual role of platelets in hemostasis andthrombogenesis is reviewed in Ruggeri, Nature Medicine, 8:1227-1234(2002).

Most steps in these processes depend on the interaction of extracellularligands with specific receptors embedded in the platelet cell membrane.In vivo, the first visible change in platelet behavior is the adhesionof platelets to an area of denuded endothelium caused by endothelialinjury. Among the micromolecular constituents which become exposed atthe denuded endothelium, collagen is considered the most reactive withplatelets. Collagen supports platelet adhesion through direct andindirect pathways and also activates platelets by initiating plateletaggregation and generating coagulant activity necessary for plugformation. Baumgartner, Thromb Haemost. 37:1-16 (1977).

The initial contact between the platelets and subendothelium involvesinteraction of the platelet glycoprotein complex GPIb-V-IX with vonWillebrand factor (vWf) bound to the exposed subendothelium. Thisinteraction appears to be a reversible process and is insufficient forstable adhesion, as illustrated by “rolling” of platelets along thevessel wall. Ruggeri, Nature Medicine, 8:1227-1234 (2002). Although thevWf interaction does not completely immobilize circulating platelets, itis essential to platelet adherence under high blood flow conditions.Subsequent irreversible binding of platelets to subendothelial collagenthrough glycoprotein GPIa-IIa (also known as integrin α₂β₁) stabilizesthe vWf interaction event, firmly anchoring the platelet to the vesselwall. Unlike vWf, collagen adhesion appears to be a slower process andis effective only under low flow conditions, or after platelets havebeen partially arrested by vWf interactions. In addition, GPIa-IIabinding induces the flattening (spreading) of platelet against thevessel wall. Spreading promotes the binding of other subendothelialadhesion factors including fibronectin, vitronectin and thrombospondin.These post-spreading interactions further stabilize the adhesion ofplatelets to the vessel wall.

GPIa-IIa is an integrin comprising an alpha and beta subunit. In theirnormal conformation, integrins have low affinity for their naturalligand but may be converted to high affinity receptors through signalsgenerated by other cell receptors Nieswandt B and Watson SP. Blood102:449-461 (2003). Stimulation of GPIa-IIa and other collagen receptorsinduces a host of physiological changes. Among these are altered cellsurface adhesion properties that result in platelet-plateletaggregation, and the secretion of various bioactive compounds. Thesecompounds include the vasoconstrictor, epinephrine, and proclottingfactors, which activate thrombin and lead to polymerization offibrinogen into the fibrin threads of a mature clot. In addition,activated platelets release ADP and thromboxane A₂ (TXA₂). Thesepowerful thrombogenic factors amplify the initial activation signal,recruiting additional platelets into the activated state.

In addition to GPIa-IIa, at least two other collagen receptors areexpressed on the platelet cell surface, namely, GPIV (CD36), and GPVI(reviewed in Farndale R W et al., J Thromb. Haemost 2:561-573, 2004).Clues to the functions of these platelet collagen receptors have comefrom the study of human patient variants. Studies of human patientvariants suggest that GPVI plays a major role in platelet-collageninteractions while contribution of GPIV remains minor. These studiesalso showed that a substantial number of individuals lacking eitherGPIa-IIa or GPVI exhibit slightly prolonged bleeding times than thosewho express GPIa-IIa or GPVI. Moreover, GPIa-IIa deficiencies generallylead to more severe bleeding disorders than those deficient in GPVI.Nevertheless, these patients rarely present the severe bleeding tendencysuch as that seen in individuals with Bernard Soulier Syndrome, which iscaused by GPIb deficiency, or Glanzmann thromasthenia, which is causedby GPIIb-IIIa deficiency.

Observations of human variants, along with recent in vitro data, suggestthat the three collagen receptors act in concert to mediatecollagen-platelet interactions. In vitro, for instance, it is nowpossible to block the activity of each collagen receptor with antibodiesspecific for the collagen receptor sites. Individually, each antibodypartially inhibits platelet adhesion to collagen and pairwisecombinations of antibodies are significantly more inhibitory,particularly when GPIa-IIa and GPVI are inhibited simultaneously.Moreover, these studies demonstrate that GPIV, GPIa-IIa, and GPVIcontribute to thrombosis through two distinct pathways, mechanisticallydistinguishable by the requirement for divalent metal cations.

Biochemical and sequence information indicates that GPIa-IIa is acation-dependent integrin-type receptor. In contrast, biochemicalstudies reveal that GPIV and GPVI do not require divalent metal cationsand are thus of the non-integrin type. Of the non-integrin class,observations of human subjects clearly suggest that GPVI is moreimportant than GPIV in the primary adhesion process. Indeed, in vitroexperiments where GPIa-IIa function is blocked by chelating divalantcations, antibodies directed against GPVI completely abolishcollagen-platelet interaction. Nakamura et al. J. Biol. Chem.273:4338-4344, (1998).

GPVI was first identified about 30 years ago by isoelectric focusing andelectrophoresis. Until recently, its function was completely undefinedand it was known merely as a platelet glycoprotein with a molecular massof approximately 62 kDa under reducing condition. However, beginningaround 1987, Dr. Minoru Okuma and associates examined several patientswith a form of thrombocytopenic purpura, a bleeding/bruising syndromecharacterized by accelerated platelet destruction and decreased numbersof circulating platelets. The platelets in some of Dr. Okuma's patientsaggregated normally in response to most agonists, including ADP,thrombin, ristocetin, and calcium ionophore (A23187) but were markedlyunresponsive to collagen. Moreover, these platelets were found to havereduced amounts, or even totally lack, the 62 kDa glycoprotein. Sugiyamaet al., Blood 69:1712-20 (1987); Moroi et al., J. Clin. Invest.84:1440-45 (1989); Ryo et al., Am. J. Hematol. 39:25-31 (1992); and Araiet al., Brit. J. Haematol. 89:124-130 (1995).

The key reagent in the early studies of GPVI function came from one ofDr. Okuma's thrombocytopenic purpura patients. This patient presentedwith massive, unexplained bleeding and was treated by transfusion withHLA-matched platelets. Subsequent detailed examination of the patient'sblood revealed a total lack of GPVI. Most surprisingly, because thispatient totally lacked GPVI, her immune system had identified the GPVImolecules on the transfused platelets as foreign antigens and producedpolyclonal antibodies against GPVI. Sugiyama et al., Blood 69:1712-20(1987).

A naturally occurring antibody is composed of two identical bindingsites, specific for a single antigenic epitope. The two antigen-specificportions are linked by a common stem, or Fc domain, to form a complexcapable of binding to two identical antigen molecules. Moreover, thedivalent nature of the antibody, in conjunction with aggregatoryproperties of the Fc domain, allow cross-linking and aggregation of manyspecific antigen molecules. Dr. Okuma found that the divalent antibodiesfrom the patient's serum caused a massive aggregation response whenmixed with normal platelets. Conversely, when the antigen-specificdomains are rendered monovalent by enzymatic removal of linking Fcdomain, the resulting Fab fragments completely abolishedcollagen-induced aggregation of normal platelets and inhibitedplatelet-collagen adhesion.

Dr. Okuma has graciously made this rare serum available to thescientific community. Unfortunately, the supply is limited, and thecircumstances surrounding its discovery are virtually irreproducible.Although the Okuma serum made possible much of the research into thefunction of GPVI and had long provided the sole method of identifying aprotein as GPVI, it has recently been discovered that the C-type lectin,convulixin, specifically binds to GPVI with high affinity and can belabeled as a probe to identify the GPVI protein. Francishetti et al.,Toxicon 35:1217-28 (1997); Polgar et al., J. Biol. Chem.272(24):13576-83 (1997); Jandrot-Perrus et al., J. Biol. Chem.272(2):27035-41 (1997). Convulxin is a venom component from the tropicalrattlesnake Crotalus durissus terrificus. In its native, divalent form,convulxin is a potent inducer of platelet aggregation and secretion ofproaggregatory and proclotting factors. The divalent nature of convulxinis critical to the aggregatory effect. Although the underlyingphysiology of the reaction is unclear, individual convulxin subunitsstill bind to GPVI, but inhibits, rather than induces aggregation. Ithas been suggested that monovalent convulxin blocks the transmission ofcollagen-induced signals to the interior of the cell.

Even more recently, the full GPVI sequence was determined. Clemetson etal., J. Biol. Chem. 274:29019-24 (1999); WO 00/68377; Jandrot-Perrus etal., Blood 96:1798-807 (2000); Ezumi et al., Biochem Biophys Res Commun.277:27-36 (2000). GPVI belongs to the immunoglobulin super family and isnon-covalently associated with the Fc receptor gamma chain (FcRγ chain).Gibbins et al., FEBS Lett. 413:255-259 (1997); Tsuji et al., J. Biol.Chem. 272:23528-23531 (1997). It is currently believed that collagenbinding to GPVI induces tyrosine phosphorylation of FcRγ. PhosphorylatedFcRγ then recruits the Syk kinase, ultimately leading to a cascade ofintracellular events including phospho-activation of Syk, andphospholipase C-γ2. These events ultimately result in increasedintercellular calcium levels and the secretion of proaggregatory andproclotting factors. Therefore, the FcRγ chain serves as thesignal-transducing part of the receptor in humans and mouse platelets.Clemetson et al., J. Biol. Chem. 274:29019-290 (1999); Jandrot-Perrus etal., Blood 96:1798-1807 (2000); Gibbins et al., FEBS Lett. 413:255-259(1997); Tsuji et al., J. Biol. Chem. 272:23528-23531 (1997).

Patients deficient in GPVI suffer from mild bleeding diathesis and theirplatelets respond poorly to collagen. Sugiyama et al., Blood69:1712-1720 (1987); Moroi et al., J. Clin. Invest. 84:1140-1445 (1989);Arai et al., Br. J. Haemtol. 89:124-130 (1995). Studies withGPVI-deficient human platelets or platelets blocked with anti-GPVI Fabfragments (obtained from a patient serum) clearly demonstrated a lack ofplatelet interaction with immobilized collagen under high and low shearrates and reduced firm adhesion to immobilized vWf under high shear.Goto et al., Circulation 106:266-272 (2002).

Studies with knockout mice deficient in the FcRγ chain, which alsoresults in a GPVI-deficient phenotype, or with mice depleted of GPVI,confirmed these observations but these animals exhibited slightlyprolonged tail bleeding time. Nieswandt et al., The EMBO Journal20:2120-2130 (2001). GPVI-deficient, FcRγ-positive mice showed bleedingtimes similar to those of wild type and GPVI-heterozygous mice.Platelets from the GPVI-deficient mice did not aggregate in response tocollagen and to convulxin, and showed dramatically reduced adhesion toimmobilized collagen under flow conditions, thereby confirming that GPVIplays a major role in collagen-induced platelet functions andthrombosis. Kato et al., Blood 102:1701-1707 (2003).

It is now accepted that GPVI is the principle receptor forcollagen-induced platelet activation, and is a critical conduit forsignal transduction. Ichinohe et al., J. Biol. Chem. 270(47):28029-28036(1995); Tsuji et al., J. Biol. Chem. 272(28):23528-31 (1997). Incontrast, the other major collagen receptor in platelets, GPIa-IIa, isprimarily involved with the cation-dependent processes for effectingstable adhesion and spreading leading to thrombus growth. Reviewed inNieswandt and Watson, Blood 102:449-461 (2003).

The need in the art for GPVI antagonists, such as antibodies againstGPVI, is highlighted by the unfortunate fact that inappropriate plateletaggregation and clot formation is a major etiologic factor in a widerange of human diseases, most commonly, vascular diseases. Excessiveplatelet deposition on the inner walls of arteries and veins contributesto atherosclerosis and arteriosclerotic plaques, which reduce the flowof blood to sensitive tissues. Ultimately, this platelet-dependentbuildup may manifest as acute myocardial infarct, chronic unstableangina, transient ischemia, stroke, peripheral vascular disease,arterial thrombosis, pulmonary embolism, restenosis, and various otherconditions.

These conditions typically begin with an abnormal clot that develops ina blood vessel, called a thrombus. Once a clot has developed, continuedflow of blood past the clot is likely to break it free from itsattachment. Such freely flowing clots are known as emboli. Emboligenerally travel through the circulation until trapped in a narrow pointin the circulatory system. This occlusion may occur in the brain, lungor coronary arteries, resulting in pain, disability or death.

Intravascular clots may result from naturally-occurring sclerosis,septicemic shock, or physical damage to blood vessels. Indeed, the veryinvasive methods used to diagnose and treat vascular disease, (e.g.vascular grafts, exploratory and in-dwelling catheters, stents, shunts,and other devices) themselves, damage vessel walls. This can activateplatelets, stimulate aggregation, and ultimately lead to the formationof thrombi and emboli, further endangering the life and health of thepatient. Thus, methods for controlling or reducing platelet aggregationand clot formation have been a long-sought goal in managing thesediseases.

SUMMARY OF THE INVENTION

The present invention provides GPVI specific antibodies that are morepotent inhibitors of collagen-induced platelet functions than thosepreviously reported in the art. Thus, the GPVI specific antibodies ofthe invention may be useful antithrombotic agents and may have reducedside effects often associated with administration of otherantithrombotic agents.

One aspect of the present invention provides a monoclonal antibodyspecific for a GPVI polypeptide, peptide, or naturally-occurring variantthereof, that inhibits collagen-induced platelet aggregation at an IC₅₀of less than about 7, 4, 3, 2, 1, 0.6, μg/ml, or any value subsumedwithin this range. The monoclonal antibody specific for a GPVIpolypeptide, peptide, or naturally-occurring variant thereof may alsoinhibit collagen-induced platelet adhesion at an IC₅₀ of less than about1, 0.5, 0.2, 0.1 μg/ml, or any value subsumed within this range.

Another aspect of the present invention provides a monoclonal antibodyspecific for a GPVI polypeptide, peptide, or naturally-occurring variantthereof, that specifically binds to a GPVI polypeptide, peptide, ornaturally-occurring variant thereof, at a Kd of equal to or lower than10⁻⁸M. In another embodiment, the anti-GPVI antibodies of the inventionspecifically bind to a GPVI polypeptide, peptide, or naturally-occurringvariant thereof, at a Kd of equal to or lower than 10⁻⁹M.

The monoclonal antibodies of the present invention also inhibitcollagen-induced ATP secretion and/or collagen-induced thromboxane A₂formation. The monoclonal antibodies include active antibody fragments.Active antibody fragments may include chemically, enzymatically, orrecombinantly produced Fab fragments, F(ab)₂ fragments, or peptidescomprising at least one complementarity determining region (CDR)specific for a GPVI polypeptide, peptide, or naturally-occurring variantthereof. In an embodiment of the invention, the CDRs comprise any one ofthe sequences of SEQ ID NOs. 1-24, or a variant thereof, wherein the CDRvariant specifically binds a GPVI polypeptide, peptide, ornaturally-occurring variant thereof. Exemplary antibodies include OM1,OM2, OM3, and OM4.

The present invention also provides a method for inhibiting plateletaggregation, collagen-induced ATP secretion, collagen-inducedthromboxane A₂ formation, and/or platelet adhesion, by contactingplatelets with a monoclonal antibody specific for a GPVI polypeptide,peptide, or naturally-occurring variant thereof.

The invention further provides a method of producing a monoclonalantibody specific for a GPVI polypeptide, peptide, ornaturally-occurring variant thereof. The method comprises immunizing aGPVI-deficient host with a GPVI antigen and obtaining the antibody. AGPVI-deficient host includes, for example, a GPVI heterozygous host anda homozygous GPVI knock-out host. Another aspect of the inventionprovides monoclonal antibodies specific for a GPVI polypeptide, peptide,or naturally-occurring variant, thereof produced by the methoddescribed.

The invention also provides an antithrombotic composition comprising apharmaceutically effective amount of a GPVI specific monoclonal antibodyof the invention. The antithrombotic agent may be used to treat apatient. Thus, an aspect of the invention provides a method of treatinga patient who, for example, is in need of treatment for vasculardisease.

The invention further provides a method for identifying antithromboticagents by contacting a GPVI antigen with a GPVI specific monoclonalantibody of the invention and a test compound, and measuring inhibitionof the binding of the monoclonal antibody to the GPVI antigen. The GPVIantigen, GPVI specific monoclonal antibody, and the test compound may beadded in any order. For example, the GPVI antigen may be contacted withthe test compound before contacted with the GPVI specific monoclonalantibody. In another example, the GPVI antigen may be contacted withGPVI specific monoclonal antibody and the test compound simultaneously.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the generation of GPVI knock-out mice.FIG. 1A shows the wild-type allele of GPVI, the targeting vector usedfor homologous recombination at exons 2 and 3, and the resulting mutantallele. FIG. 1B shows the size differences of the 5′ and 3′ fragmentsderived by cleavage with restriction enzymes in wild-type and mutantgenomes.

FIG. 2 is a bar graph showing the effects of Fab fragments of OM1, OM2,OM3, and OM4 at 0.1-100 μg/ml on platelet (human) adhesion to fibrillarcollagen under static conditions.

FIG. 3 is a bar graph showing the effects of Fab fragments of OM1, OM2,OM3, and OM4 at 0.001-1 μg/ml on Mg²⁺-independent (GPVI-dependent) humanplatelet adhesion to fibrillar collagen under static conditions.

FIG. 4 illustrates the effects of Fab fragments of OM1, OM2, OM3, OM4,and REOPRO® on platelet (human) adhesion to acid insoluble collagenunder high shear stress (2600 sec⁻¹) conditions.

FIG. 5 is a Western blot of the OM series antibodies (OM1, OM2, OM3, andOM4) and convulxin (CVX) reacting with GPVI in human platelet lysate.

FIG. 6 illustrates the complete lack of platelet aggregation induced bycollagen and convulxin in GPVI knock-out animals.

FIG. 7 illustrates the lack of interaction of platelets from GPVIknock-out mice to acid insoluble collagen under high shear stressconditions.

FIG. 8 is a graph showing the effect of the OM2 Fab fragment and REOPRO®on ex vivo collagen-induced platelet aggregation in Cynomolgus monkeys.

FIG. 9 is a graph showing the effect of the OM2 Fab fragment andREPOPRO® on skin bleeding time in Cynomolgus monkeys.

FIG. 10 is a graph showing the anti-aggregation effect of the OM2 Fabfragment and REPOPRO® over time after bolus injection (0.4 mg/kg) inCynomolgus monkeys.

FIG. 11 is a graph showing the time-course effect of the OM2 Fabfragment and REPOPRO® on skin bleeding time after bolus injection (0.4mg/kg) in Cynomolgus monkeys.

FIG. 12 illustrates the effects of the OM4 Fab and 7E3 F(ab′)₂ fragmentson ex vivo collagen-induced platelet aggregation in rats.

FIG. 13 illustrates the effects of the OM4 Fab and 7E3 F(ab′)₂ fragmentson bleeding time in rats. FIG. 13A shows the effects on nail bleedingtime and FIG. 13B shows the effects on tail bleeding time.

FIG. 14 illustrates the effects of the OM4 Fab and 7E3 F(ab′)₂ fragmentson platelet count in rats.

FIG. 15 is a bar graph showing the effect of the OM4 Fab fragment onarterial thrombus formation in rats when administered before (FIG. 15A)and after (FIG. 15B) endothelial injury.

FIG. 16 is a graph showing the concentration-dependent binding ofbiotinylated OM2 Fab fragment to human platelets in vitro.

DESCRIPTION OF THE EMBODIMENTS

This invention describes novel GPVI specific antibodies that are potentinhibitors of collagen-induced platelet responses, including, but notlimited to, platelet aggregation, adhesion, collagen-induced ATPrelease, and thromboxane A₂ (TXA₂) formation. The invention alsodescribes methods for producing the anti-GPVI antibodies. The anti-GPVIantibodies of the invention may be useful for inhibiting thrombusformation and for treating patients in need of anti-thrombotictreatment.

The term “antibodies” includes monoclonal antibodies. The monoclonalantibodies of the invention include active antibody fragments, such asF(ab′)₂, and Fab fragments, as well as any recombinantly producedbinding partners. Antibodies are defined to be “specifically binding” ifthey bind a GPVI polypeptide, peptide, or naturally-occurring variantthereof, with a dissociation constant (Kd) equal to or lower than 10⁻⁷M.In an embodiment of the invention, the anti-GPVI antibodies specificallybind to a GPVI polypeptide, peptide, or naturally-occurring variantthereof, at a Kd of equal to or lower than 10⁻⁸M. In another embodiment,the anti-GPVI antibodies of the invention specifically bind to a GPVIpolypeptide, peptide, or naturally-occurring variant thereof, at a Kd ofequal to or lower than 10⁻⁹M. Affinities of binding partners orantibodies may be readily determined using conventional techniques, forexample by measuring the saturation binding isotherms of ¹²⁵I-labeledIgG or its fragments, or by homologous displacement of ¹²⁵IgG byunlabeled IgG using nonlinear-regression analysis as described byMotulsky, in Analyzing Data with GraphPad Prism (1999), GraphPadSoftware Inc., San Diego, Calif. Other techniques are known in the art,for example, those described by Scatchard et al., Ann. NY Acad. Sci.,51:660 (1949). GPVI polypeptides, peptides, or naturally-occurringvariants thereof, are described in U.S. Publication No. 2003/0186885,and is incorporated herein by reference in its entirety.

Antibodies may be readily generated from a variety of sources, forexample, horses, cows, goats, sheep, dogs, chickens, rabbits, mice,hamsters, or rats, using procedures that are well-known in the art. Inan embodiment of the invention, the host animals are Armenian hamsters.In another embodiment, the host animals are GPVI-deficient animals. Asused herein, “GPVI-deficient” refers to about 50% or greater reductionin endogenous GPVI production in an animal compared to a wild-typeanimal. The reduction in endogenous GPVI production may be such thatGPVI production is completely inhibited. GPVI-deficient animals may begenerated by a number of methods known in the art. These may includemanipulation of GPVI production at the nucleic acid (DNA or RNA) levelin an animal. GPVI-deficient animals may be generated by methodsincluding, but not limited to, knock-out (see, e.g., Galli-Taliadoros etal., J. Immunol. Methods 181:1-15, 1995; Robbins, Circ. Res. 73:3-9,1993; Hergueux et al., Transplant Proc. 25:30-32, 1993), knock-in(Colucci-Guyon et al., Cell 79:679-694, 1994; Le Mouellic et al., PNAS87:4712-4716, 1990; Hanks et al., Science 269:679-682, 1995; Wang etal., Nature 379:823-825, 1996), mutation (Askew et al., Mol. Cell. Biol.13:4115-4124, 1993; Stacey et al., Mol. Cell. Biol. 14:1009-1016, 1995;Hasty et al, Nature 350:243-246, 1991; Valancius et al, Mol. Cell. Biol.11:1402-1408, 1991; Wu et al., PNAS 91:2819-2823, 1994; Horie et al.,Gene 166:197-204, 1995; Toth et al., Gene 178:161-168, 1996), deletion(You et al., Nature Genet., 15:285-288, 1997; Holdener-Kenny et al.,Bioessays 14:831-839, 1992), antisense oligonucleotide technology(Wagner et al., Nature Biotechnol. 14:840-844, 1996; Kitajima et al.,Science 258:1792-1795, 1992; Urban et al., Farmaco. 58:243-58, 2003;Orum et al., Curr Opin Mol Ther. 3:239-43, 2001; Sohail et al., CurrOpin Mol Ther. 2:264-71, 2000; Smith et al., Eur J Pharm Sci. 11:191-8,2000), interfering RNA (RNAi) technology (Scherr et al., Curr Med. Chem.10:245-56, 2003; Nishikura, Cell 107:415-418, 2001; Hannon, Nature4418:244-251, 2002; U.S. Pat. No. 5,506,559), or by using any otherchemicals, naturally-occurring, recombinant, or synthetic peptides,polypeptides, proteins, polysaccharides, small molecules and othercompounds designed to reduce or inhibit GPVI production in a host.

Without being bound to theory, hosts that produce none or lower thannormal amounts of endogenous GPVI may mount a stronger immune reactionto GPVI than those that make normal levels of GPVI. Thus, antibodies toGPVI that are more effective at inhibiting collagen-induced plateletresponses such as platelet aggregation, thrombogenesis, and/or plateletactivation at lower doses may be produced compared to those obtainedfrom normal hosts that make GPVI. Methods for producing antibodies inknock-out animals have been described in Pass et al., Scand. J. Immunol.58:298-305 (2003); Zlot et al., J. Lipid Res. 40:76-84 (1999); Declercket al., J. Biol. Chem. 270:8397-8400 (1995); and Castrop et al.,Immunobiology 193:281-287 (1995).

Hosts may be immunized as described in US Patent Publication No. US2003/0186885 A1, which is herein incorporated by reference in itsentirety. Briefly, hosts may be immunized with “GPVI antigen” whichincludes, but is not limited to, native GPVI polypeptides, peptides, ornaturally-occurring variants thereof, isolated from platelets or otherGPVI-expressing cells; recombinant GPVI polypeptides, peptides, orrecombinant forms of naturally-occurring variants thereof, expressedfrom prokaryotic or eukaryotic cells; platelets obtained from variousspecies, including human; cells expressing GPVI polypeptides, peptides,or naturally-occurring variants thereof; nucleic acids encoding GPVIpolypeptides, peptides, or naturally-occurring variants thereof; or anycombination thereof.

Purified GPVI polypeptides, or a peptide based on the amino acidsequence of GPVI polypeptides conjugated to an adjuvant or carrier, aretypically administered to the host animal intraperitoneally. Theimmunogenicity of GPVI polypeptides may be enhanced through the use ofan adjuvant, for example, Freund's complete or incomplete adjuvant.Following booster immunizations, small samples of serum are collectedand tested for reactivity to GPVI polypeptides. Examples of variousassays useful for such determination include those described in:Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radioimmunoprecipitation (RIP), enzyme-linked immuno-sorbent assays(ELISA), dot blot assays, and sandwich assays and FACS. See U.S. Pat.Nos. 4,376,110 and 4,486,530.

Monoclonal antibodies can be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. No.RE 32,011, U.S. Pat. Nos. 4,902,614, 4,543,439, and 4,411,993;Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980.Briefly, the host animals are injected intraperitoneally at about 1 weekintervals with a GPVI antigen, optionally in the presence of adjuvant.Immunizations are carried out until desired titer of antibody isachieved.

Mouse sera are then assayed for antibody titer by FACS analysis usingGPVI-FcRγ chain transfected CHO cells, or any other method known in theart. The selected mice are given a booster dose of the GPVI antigen.Three days later, the mice are sacrificed and their spleen cells arefused with commercially available myeloma cells, P3U1 (ATCC), followingestablished protocols. Myeloma cells are washed several times inserum-free media and fused to mouse spleen cells. The fusing agent is50% PEG (Roche). Fusion is plated out into eight 96-well flat bottomplates (Corning) containing HAT supplemented DMEM media and allowed togrow for 1-2 weeks. Supernatants from resultant hybridomas are collectedand analyzed for the presence of anti-GPVI antibodies by performing FACSanalysis using CHO cells expressing GPVI and FcRγ-chain. FACS analysisis also performed using wild-type CHO cells to eliminate clonesproducing antibodies against CHO cell antigens. Positive clones can begrown in bulk culture and supernatants are subsequently purified over aProtein A or G Sepharose column (Pharmacia). It is understood that manytechniques could be used to generate antibodies against GPVIpolypeptides and peptides and that this embodiment in no way limits thescope of the invention.

The monoclonal antibodies of the invention may be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, in Strategies in Molecular Biology 3:1-9 (1990), which isincorporated herein by reference. Similarly, binding partnersconstructed, for example, using recombinant DNA techniques toincorporate the variable regions of a gene that encodes a specificbinding antibody, are included in the monoclonal antibodies of theinvention. Such a technique is described in Larrick et al.,Biotechnology, 7:394 (1989).

Other types of antibodies may be produced in conjunction with the stateof knowledge in the art. For example, antiidiotype antibodies may beobtained by immunizing a host with an antigen comprising the antigenbinding site of a purified monoclonal anti-GPVI antibody, and testingthe resultant sera or monoclonal supernatant for activity as describedin Knight et al., Mol. Immunol. 32:1271-81 (1995). In an embodiment ofthe invention, the antiidiotype antibodies may be obtained by immunizinga host with a peptide comprising a complementarity determining region(CDR) of an anti-GPVI antibody of the invention. The antiidiotypeantibodies of the invention include active antiidiotype antibodyfragments, which refer to chemically, enzymatically, or recombinantlyproduced fragments of an antiidiotype antibody, including, Fab, F(ab)₂,or peptides comprising at least one complementarity determining region(CDR) that bind specifically to an anti-GPVI antibody. In addition, theinvention comprises biosynthetic GPVI antibody binding sites, asdescribed by Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 (1988);single-domain antibodies comprising isolated heavy chain variabledomains, as described by Ward et al., Nature 341:544 (1989); andantibodies that have been engineered to contain elements of humanantibodies that are capable of specifically binding GPVI polypeptides.Anti-GPVI antibodies may also be generated using the phage displaytechnology as described in Ventor et al., Ann Rev. Immunol. 12:43355(1994) and the references cited therein, all of which are incorporatedherein by reference.

The antibodies of the invention also include active antibody fragments,which refer to chemically, enzymatically, or recombinantly producedfragments of an antibody, including, Fab, F(ab)₂, or peptides comprisingat least one complementarity determining region (CDR) that bindspecifically to a GPVI polypeptide, peptide, or a naturally-occurringvariant thereof. A common enzymatic method utilizing pepsin or papainremoves the Fc antibody domain to produce bivalent F(ab)₂ and monovalentFab fragments. These procedures are basically described in Gorini etal., J. Immunol. 103:1132 (1969); Handbook of Experimental ImmunologyVol 1: DM Wier (ed), Blackwell Alden Press, Oxford, UK, 1997; andAntibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; and U.S. Pat. No. 4,470,925(Auditore-Hargreaves), all of which are incorporated herein byreference.

Intact GPVI-specific antibodies, and antibody fragments, such as Fab andF(ab)₂ fragments, may be covalently coupled to drugs or carriermolecules. In addition, the GPVI-specific antibodies of the inventionmay be cross-linked, directly, or through a suitable carrier molecule,to form multivalent complexes. In one embodiment, F(ab)₂ fragments aremetabolically stabilized by covalent cross linking as described in Renoet al. (U.S. Pat. No. 5,506,342) (incorporated herein by reference)

Monoclonal antibodies specific for a GPVI polypeptide, peptide, or anaturally-occurring variant thereof, may be tested for their ability toblock platelet activation by ligand (collagen)-dependent binding.Monoclonal antibodies which block platelet functions may be usefulantithrombotic agents.

The antibodies of the present invention may also be humanized. Human andhumanized antibodies are thus preferred for clinical use. See, forexample, LoBugio et al., Proc. Natl. Acad. Sci. USA 86:4220-24 (1989);Meredith et al., J. Nucl. Med. 33, 23-29 (1992); Salah et al., Hum.Antibod. Hybridomas 3:19-24 (1992); Knight et al., Mol. Immunol.32:1271-81 (1995); and Lockwood et al., Q. J. Med. 89:903-12, (1996).

Development of fully human antibodies generally require a suitablesource of human immune B lymphocytes. One method for generating humanantibodies involves the immunization and expansion of B lymphocytes withsuitable specificities from pools of naïve B cells obtained fromnon-immunized individuals that were placed in in vitro culture. Ohlinand Borrebaeck, in Methods of Immunological Analysis, vol. II, Masseyeffet al. (eds), VCH Verlagsgesellschaft mbH, Weinheim, p. 298-325 (1992);Borrebaeck and Ohlin, in Protocols in Cell and Tissue Culture, Doyle etal. (eds), J. Wiley & Sons Ltd., Chichester 25E:1.1-7 (1993). Humanantibodies against HIV-1 glycoproteins have been developed by thismethod. Ohlin et al., Immunology 68:325-331 (1989); Ohlin et al., Clin.Exp. Immunol. 89:290-295 (1992); Duenas et al., Immunology 89:1-7(1996). More recently, in vivo technologies have been developed thatutilize animals either engrafted with human immune cells or those thathave been introduced with the entire human immunoglobulin loci. IIan etal., Curr. Opin. Mol. Ther. 4:102-109 (2002); Ishida et al., CloningStem Cells 4:91-102 (2002). Fully human antibodies have been producedupon immunization of these animals with various human antigens.

Humanized antibodies may be generated by replacing most, or all, of thestructural portions of a monoclonal antibody with corresponding humanantibody sequences. Consequently, a hybrid molecule is generated inwhich only the antigen-specific variable, or complementarity determiningregion (CDR) is composed of non-human sequence. Various strategies fordesigning humanized antibodies are reviewed in Winter and Milstein,Nature 349:293-99 (1991); Harris, BCSTBS5 23(4):1035-38 (1995); Morrisonand Schlom, in Important Advances in Oncology, J. B. Lippincott Co.(1990); L. Presta, “Humanized Monoclonal Antibodies,” in Annual Reportsin Medicinal Chemistry, Academic Press, (1994); and A. Lewis and J.Crowe, “Generation of Humanized Monoclonal Antibodies by ‘Best Fit’Framework Selection and Recombinant Polymerase Chain Reaction” inGeneration of Antibodies by Cell and Gene Immortalization. Year Immunol.1993, vol 7, pp 110-118, (C. Terhorst, F. Malvasi, and A. Albertini(eds.) Basel, Karger, each of which is incorporated herein by reference.

Antibodies specific for a GPVI polypeptide, peptide, ornaturally-occurring variant thereof, may also be humanized by selectingand purifying anti-GPVI antibodies by Ig-specific adsorption, such asProtein A chromatography, or by affinity chromatography usingimmobilized GPVI peptides. The heavy and light chains may be dissociatedby standard means, and the individual chains purified. A partial aminoacid sequence of the individual chains may be determined and degenerateoligonucleotides may be generated for each chain according to the methodof Lathe et al., J. Mol. Biol. 183:1-12 (1985). The DNA encoding theseantibody chains may then be cloned and sequenced from the anti-GPVIantibody-producing cell by PCR or other standard methods.

The antibody DNA and amino acid sequence may be analyzed and comparedwith known sequences of human heavy and light chains. Based on thesequence comparisons, the GPVI-specific antibody chains may be humanizedby replacing portions of the non-human DNA with human sequences, thusforming a chimeric antibody with specificity to GPVI. In one embodiment,the GPVI-specific antibody is humanized with human J1 and K constantregions using the expression vectors described by Sun et al., Proc.Natl. Acad. Sci. USA 84:214-218 (1987). Methods for the preparation ofnonhuman-human hybrids are well known in the art and described in detailin, for example, Knight et al., Mol. Immunol. 32:1271-81 (1995); U.S.Pat. No. 5,705,154 (Dalie et al.); 5,693,322 (Creekmore et al.); U.S.Pat No. 5,677,180 (Robinson et al.); U.S. Pat. No. 5,646,253 (Wallace etal.); U.S. Pat. No. 5,585,097 (Bolt et al.); U.S. Pat. No. 5,631,349(Diamantstein et al.); and U.S. Pat. No. 5,580,774 (Beavers et al.)(each of which is incorporated herein by reference). To maximize theproduction of high affinity chimeric antibodies, the methods of Queen etal., (U.S. Pat. No. 5,585,089) and Queen et al., Proc. Nat. Acad. Sci.USA, 86:10029-33 (1989), may be employed.

Humanized antibodies may also be generated using the phage displayapproach as taught in Rader et al., Proc. Nat. Acad. Sci. USA,95:8910-8915 (1998) and Steinberger et al., J. Biol. Chem.275:36073-36078 (2000) and as exemplified by Son et al., J ImmunolMethods. 286:187-201 (2004), Lee et al., J Immunother. 27:201-210(2004), and by others skilled in the art.

The part of the antibody molecule that binds to an antigen is comprisedof only a small number of amino acids in the variable (V) regions of theheavy (VH) and light (VL) chains. These amino acids are brought intoclose proximity by folding of the V regions. Comparisons of the aminoacid sequences of the variable regions of IgG show that most of thevariability resides in three regions called the complementaritydetermining regions (CDRs). Each chain (H and L) contains three CDRs.Antibodies with different specificities have different CDR's whileantibodies of the exact same specificity generally have identical orhighly conserved CDR's. The present invention encompasses monoclonalantibodies or peptides comprising at least one complementaritydetermining region (CDR), or a variant thereof, of the GPVI antibodiesof the invention. The invention encompasses monoclonal antibodies orpeptides comprising at least one of the amino acid sequences of SEQ IDNOs. 1-24, or variants thereof.

A “variant” of an antibody or a peptide comprising a CDR herein refersto an antibody or peptide comprising an amino acid sequencesubstantially identical to SEQ ID NOs. 1-24, but which has an amino acidsequence different from that of SEQ ID NOs. 1-24 because of one or moredeletions, insertions or substitutions. The variant also retains atleast 70, 80, 90 or 100% of its binding affinity to a GPVI polypeptide,peptide, or naturally-occurring variant thereof, compared with anantibody or peptide comprising its corresponding CDR. Binding affinitiesmay be determined according to any method known in the art, for example,as taught by Fujimura et al., Thromb. Haemost. 87:728-734 (2002) and asexemplified in Example 4 below. A variant comprises a CDR that ispreferably at least 60%, 65%, 70%, 80%, 85%, or 90% identical to SEQ IDNOs. 1-24. The percent identity can be determined, for example, bycomparing sequence information using the GAP computer program, version6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess (Nucl. Acids Res.14:6745, 1986), as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Variants may comprise conservatively substituted sequences. Conservativesubstitution refers to replacement of a given amino acid residue with aresidue having similar physiochemical characteristics. Examples ofconservative substitutions include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another, orsubstitutions of one polar residue for another, such as between Lys andArg; Glu and Asp; or Gln and Asn. Other such conservative substitutions,for example, substitutions of entire regions having similarhydrophobicity characteristics, are well known.

Candidate anti-GPVI antibodies may be screened for effects on plateletadhesion and activation using various assays known in the art. Theseinclude, for example, the platelet adhesion inhibitor assay described inU.S. Pat. No. 5,686,571; a modified constant flow assay of Diaz-Ricartet al. (Blood 82:491-496, 1993) that allows the use of a smaller volumeof blood and less antibody, as described in Brown and Larson (BMCImmunology 2:9-15, 2001); the plate assay described in Matsuno et al.(British J. Haematology 92:960-967, 1996) and in Nakamura et al. (J.Biol. Chem. 273(8):4338-44, 1998) (the “Nakamura procedure”), (each ofwhich is specifically incorporated herein by reference). In each case,candidate GPVI agonists or antagonists may be pre- or co-incubated withthe reaction components in the presence or absence of Mg²⁺. Incubationin the absence of Mg²⁺ blocks the function of GPIa/IIa such that theremaining collagen-dependent activity is primarily mediated by the GPVIreceptor.

A modified Nakamura procedure may be used to measure platelet adhesionto immobilized acid insoluble fibrillar collagen under staticconditions. The modified Nakamura assay is described briefly below. Themajor modification to the original assay includes the replacement of⁵¹Cr-labeled platelets with unlabeled platelets and the measurement ofadhesion by quantification of LDH activity released by adherentplatelets using a commercially available kit. One of skill in the artrecognizes how to make other modifications to the assay conditions inview of the particular anti-GPVI antibody tested.

Adhesion Assay—Microtiter wells are coated with type I acid-insolubleequine tendon fibrillar collagen. Platelets at a concentration of4×10⁸/ml are suspended in Tyrode-HEPES buffer or in Tyrode-HEPES buffersupplemented with Mg²⁺ (1 mM), and adhesion assays are carried out asdescribed previously (Tandon et al., Br. J. Haematol. 89:124-30, 1995).Briefly, the platelets are incubated with a sample such as an antibodysolution for 30 minutes at room temperature prior to their addition tothe collagen-coated wells. Adhesion is carried out for 60 minutes atroom temperature in the presence and absence of Mg²⁺. Unattachedplatelets are removed by repeated washing of the wells and the adheredplatelets are solubilized in Triton X-100. A commercially available LDHmeasuring kit (CytoTox 96, Promega, Madison, Wis., USA) based on acalorimetric assay is used to measure released LDH activity.

Assay for ATP Release and Thromboxane A₂ (TXA₂)generation—Collagen-induced ATP release is measured in a dual channellumiaggregometer (Model 650CA—Chronolog Corporation Havertown Pa., USA).Briefly, platelet rich plasma (platelet count adjusted to 3×10⁸/ml withplatelet poor plasma) is mixed with luciferase-luciferin reagent(Chronolog Corporation). Platelets are incubated at 37° C. for 5 minutesin the presence and absence of a test antibody solution, e.g. Fabfragments, prior to challenge with collagen. Aggregation and ATP releaseare measured simultaneously. At desired times, the reaction is stoppedby addition of a cocktail of inhibitors that inhibit synthesis of TXA₂.The supernatant of platelet suspension is transferred to a small tubeand frozen at −20° C. until measured for collagen-induced TXA₂formation. TXA₂ is measured as TXB₂, a stable metabolite of TXA₂.

Platelet Aggregation Assay—A simple assay for detecting or determiningantithrombotic activity is provided by the platelet aggregation assaydescribed in Sun et al., J Cardivascular Pharmcol. 40:557-585 (2002).Anti-GPVI antibodies, e.g., intact IgG, F(ab′)₂, or Fab fragments, orcontrol buffer (0.15 M NaCl, 0.01 M Tris.HCl, pH 7.4), is added to acuvette containing platelet-rich plasma (200 μl). The mixture isincubated for 3 to 5 minutes at 37° C. in the heating module of anaggregometer prior to inducing aggregation with collagen. The cuvette isplaced in a four channel aggregometer (AG10 Kowa, Japan), which measuresthe kinetics of particle formation by laser scattering and aggregationby changes in light transmission. Aggregation is initiated with 0.5-4μg/ml of collagen. The optimal concentrations or collagen are those thatgive at least 70% change in light transmission and are determined foreach experiment. Aggregation is monitored for at least 8-10 minutesafter the addition of collagen.

In-vitro Assay—The GPVI specific antibodies or antibody fragments may befurther assayed using the systems developed by Diaz-Ricart andco-workers (Arteriosclerosis, Thromb. Vasc. Biol. 16:883-888, 1996).This assay determines the effect of GPVI antibodies on platelets underflow conditions using de-endothelialized rabbit aorta and humanendothelial cell matrices.

In vivo Assay—The in vivo activity of GPVI antibodies or antibodyfragments may be assayed using standard models of platelet function asdescribed in Coller and Scudder, Blood 66:1456-59 (1985); Coller et al.,Blood 68:783-86 (1986); Coller et al., Circulation 80:1766-74 (1989);Coller et al., Ann. Intern. Med. 109:635-38 (1988); Gold et al.,Circulation 77, 670-77 (1988); and Mickelson et al., J. Molec. CellCardiol. 21:393-405 (1989).

The above tests demonstrate that the GPVI specific antibodies of theinvention are more potent than those previously reported by others.Specifically, the GPVI specific antibodies of the invention inhibitcollagen-induced platelet aggregation at a lower IC₅₀ than antibodies inthe art. The term “IC₅₀” is known in the art as the concentration atwhich 50% inhibition is observed and is any positive value greater thanzero. The IC₅₀ for inducing inhibition of collagen-induced plateletaggregation was determined using a concentration of collagen thatinduced 70-90% platelet aggregation within 5 minutes of its contact withplatelets. The terms “inhibition” or “inhibit” refers to a decrease orcessation of any phenotypic characteristic or to the decrease orcessation in the incidence, degree, or likelihood of thatcharacteristic. In the context of platelet aggregation, “inhibition”refers to a measurable decrease or cessation in the aggregation ofplatelets. Such inhibition may be detected by the test described above,or by any other method known in the art. Similarly, “inhibition” in thecontext of platelet adhesion refers to a measurable decrease orcessation in the adhesion of platelets to a surface and such inhibitionmay be detected by the test described above, or by any other methodknown in the art.

The GPVI specific antibodies of the invention inhibit collagen-inducedplatelet aggregation at an IC₅₀ of less than about 7, 4, 3, 2, 1, 0.6μg/ml, or any value subsumed within this range. The GPVI specificantibodies of the invention also inhibit collagen-induced plateletadhesion at an IC₅₀ of less than or equal to about 1, 0.5, 0.2, 0.1μg/ml, or any value subsumed within this range. The GPVI specificantibodies of the invention also inhibit collagen-induced ATP secretionand/or collagen-induced thromboxane A₂ formation. The GPVI specificantibodies of the invention include active antibody fragments. Activeantibody fragments include chemically, enzymatically, or recombinantlyproduced Fab fragments, F(ab)₂ fragments, and peptides comprising atleast one complementarity determining region (CDR) specific for a GPVIpolypeptide, peptide, or naturally-occurring variant thereof.

The GPVI specific antibodies of the invention may be formulated intopharmaceutical compositions according to known methods. Thepharmaceutical composition of the invention comprises at least one GPVIspecific antibody and include, but are not limited to, intact monoclonalantibodies; Fab fragments; F(ab)₂ fragments; and peptides comprising atleast one CDR sequence, or variant thereof. The GPVI specific antibodymay be combined with other known active materials.

Compositions of the invention include at least one GPVI specificantibody admixed with pharmaceutically acceptable excipients, including,but not limited to, diluents (e.g., Tris-HCl, acetate, phosphate,water), preservatives (e.g., Thimerosal, benzyl alcohol, parabens),emulsifiers, salts, polymers, buffers, solubilizers, adjuvants and/orcarriers. Suitable excipients and their formulations are described inRemington: The Science and Practice of Pharmacy, 20th ed, MackPublishing Co. (2000). In addition, such compositions may contain GPVIspecific antibodies complexed with polyethylene glycol (PEG), metalions, incorporated into polymeric compounds such as polyacetic acid,polyglycolic acid, hydrogels, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar, multilamellar, or colamellarvesicles, erythrocyte ghosts or spheroblasts.

The instant invention also comprises methods of inhibiting thrombosis,for example, by inhibiting platelet aggregation or platelet adhesion,comprising contacting activated or resting platelets with antibodiesdirected against GPVI. “Inhibiting thrombosis” refers to a decrease orcessation of a thrombotic event or to the decrease in the incidence,degree, or likelihood of thrombotic events in a patient, patientpopulation, or in vitro test systems. The invention also relates to thetreatment of a patient, hereby defined as any person or non-human animalin need of anti-thrombotic treatment to reduce the incidence,likelihood, or degree of thrombosis, or platelet aggregation, orplatelet activation, or to any subject for whom treatment may bebeneficial for the treatment of vascular disease, including humans andnon-human animals. Such non-human animals to be treated include alldomesticated and feral vertebrates including, but not limited to: mice,rats, rabbits, fish, birds, hamsters, dogs, cats, swine, sheep, horses,cattle, and non-human primates.

The treatment of a patient comprises the administration of apharmaceutically effective amount of an anti-GPVI antibody-containingcomposition of the invention. One of ordinary skill in the art mayempirically determine the optimum dosage and dosage schedule foradministering these compositions. Nevertheless, a pharmaceuticallyeffective amount is that amount which provides a measurableanti-thrombotic effect, for example, a reduction in the incidence,degree, or likelihood of thrombosis, platelet aggregation, or plateletactivation as measured in vivo or in vitro, or provides a measurabledecrease in the likelihood, incidence, or degree of vascular disease,clot or emboli formation, or ischemic events in a patient.

A pharmaceutically effective amount may be administered as a single doseor as multiple doses over the course of treatment. A kit within thescope of the invention comprises encompasses a container containing oneor more doses of a pharmaceutically effective amount of an anti-GPVIantibody-containing composition of the invention. Such kits encompassanti-GPVI antibodies alone, admixed or suspended with a suitablepharmaceutically acceptable diluent and/or other excipient, orformulated to be admixed or suspended in a suitably acceptable diluentand/or other excipient prior to administration.

The compositions of the invention may be administered by any methodfamiliar to those of ordinary skill in the art, for example, intravenousadministration by bolus injection, continuous, or intermittent infusion.In alternative embodiments, the compositions may be administeredintraperitoneally, intracorporeally, intra-articularly,intraventricularly, intrathecally, intramuscularly, subcutaneously,topically, tonsillarly, mucosally, intranasally, transdermally,intravaginally, orally, or by inhalation.

The GPVI specific antibodies of the invention may also be used to screenfor compounds that may be useful as anti-thrombotic agents. Thescreening method comprises contacting a GPVI antigen with a testcompound and a GPVI specific antibody and measuring the inhibition ofbinding of the GPVI specific antibody to the GPVI polypeptide, peptide,or a naturally-occurring variant thereof. The GPVI antigen, GPVIspecific antibody, and the test compound may be added in any order. Forexample, the GPVI antigen may be contacted with the test compound beforecontacted with the GPVI specific monoclonal antibody. In anotherexample, the GPVI antigen may be contacted with GPVI specific monoclonalantibody and the test compound simultaneously.

Inhibition of binding suggests that the test compound competes for, orotherwise interferes with, the same binding site on GPVI as the GPVIspecific antibody. “GPVI antigen” in the context of screening foranti-thrombotic agents, refers to, but is not limited to, native GPVIpolypeptides, peptides, or naturally-occurring variants thereof,isolated from platelets or other GPVI-expressing cells; recombinant GPVIpolypeptides, peptides, or naturally-occurring variants thereof,expressed from prokaryotic or eukaryotic cells; or cells expressing GPVIpolypeptides, peptides, or naturally-occurring variants thereof. A testcompound may be any chemical, protein, peptide, polypeptide, or nucleicacid (DNA or RNA). The test compound may be naturally-occurring or maybe synthesized by methods known in the art. The screening method of theinvention may employ high-throughput screening (HTS) methods.High-throughput screening methods are reviewed in Khandurina et al.,Curr Opin Chem Biol. 6:359-66 (2002); Kumble, Anal Bioanal Chem.377:812-819 (2003); and Bleicher et al., Nature Rev Drug Disc 2:369-378(2003).

A compound identified as inhibiting the binding of a GPVI specificantibody, or an antibody fragment thereof, to a GPVI antigen, may befurther tested for its effect on platelet functions by any of themethods disclosed herein, or by other methods known in the art. Theseplatelet functions include collagen-induced platelet aggregation,collagen-induced platelet adhesion, collagen-induced ATP secretion, andcollagen-induced thromboxane A₂ formation.

The present invention is illustrated by the following Examples, whichare not intended to be limiting in any way.

EXAMPLE 1 Preparation of Monoclonal GPVI Antibodies

Normal mice (Balb/c, female), Armenian hamsters (male) and GPVIknock-out mice (produced at Otsuka GEN institute) were immunized toproduce monoclonal antibodies as described below.

GPVI knockout mice were generated as previously described (Mori et. al.Neurosci. Res. 43: 251-7, 2002). The targeting vector was constructed byreplacing a genomic fragment of the GPVI gene (Ezumi Y. et al., BiochemBiophys Res Comm 277:27-36, 2000) from 129/Sv mouse genomic λ clonescontaining the last 5 bases of exon 2 to the first half of exon 3(Clalsite) with the pMC1-neo-polyA (Stratagene) cassette as shown inFIG. 1A. The linearized construct was electroporated into AB2.2 ES cellsderived from 129/Sv mouse (Lexicon Genetics Inc., The Woodlands, Tex.)and the cells were selected in G-418. G-418-resistant ES cell cloneswere screened for successful homologous recombination at exons 2 and 3by probing SphI- or KpnI-digested genomic DNAs with 5′ or 3′ externalprobes, respectively (FIG. 1B; Southern blotting data not shown).Chimeric mice derived from the homologous recombinant ES cells weremated with C57BU6J mice to obtain heterozygous mutants (F1). Homozygousmutants (F2) were derived by mating the obtained heterozygous mutantsand confirmed by Southern blotting. Mice were genotyped by polymerasechain reaction (PCR) using genomic DNA extracted from tail snips. Noabnormalities in birth rate, birth weight, growth and development,Mendelian distribution, or bleeding disorders were observed in thehomozygous mutants. Background-matched wild-type and heterozygous micewere used as controls. Armenian hamsters were obtained from CytogenResearch and Development Inc. Boston, Mass. All animals were kept andbred according to the Institutional Animal Care and Use Committee(IACUC) protocol at Otsuka Maryland Medicinal Laboratories.

Normal mice were immunized with plasmid containing GPVI cDNA(“p-target”), CHO cell line expressing GPVI-FcRγ chain (“CGP6”, whereintransfection was performed using Lipofectamine™ 2000 from Invitrogen),GPVI purified from GPVI-FcRγ expressing CHO cells (“PGP6”), native GPVIpurified from human platelets (“nGP6”) and recombinant partial GPVI(lacking the first Ig domain) expressed in E. coli (“PAGP6”). NativeGPVI from human platelets and PGP6 from GPVI-FcRγ transfected cells werepurified by combining the lectin affinity, ion-exchange chromatography,and convulxin-affinity methods described in U.S. Publication No.2003/0186885.

CHO cells stably expressing GPVI and FcRγ (“CGP6”) were established byco-transfection of pTarget vector (Promega) containing full-length humanGPVI cDNA (“p-target”) and pcDNA3.1 (+)zeocin vector (Invitrogen)containing full-length FcRγ cDNA using Lipofectamine 2000 (Invitrogen).Cells expressing both receptors were selected in medium supplementedwith G418 and zeocin. Expression of GPVI was detected by FACS analysiswith the Epic Altra FACS analyzer (Beckman Coulter) using a polyclonalhuman anti-GPVI antibody (Dr. Okuma's serum described earlier) or aFITC-labeled convulxin. Detection of FcRγ expression was performed byimmunoblotting using commercially available anti-FcRγ polyclonalantibody (Upstate Biotechnology).

Recombinant partial GPVI was prepared by inserting into the pET21 vector(Novagen) a cDNA encoding a GPVI polypeptide lacking the entire first Igdomain of human GPVI. This partial protein was expressed in E. Colistrain BL21 (DE3). Expressed protein (“PAGP6”) was purified from theinclusion bodies as described by the manufacturer.

Armenian hamsters were immunized with CGP6 and human platelets. GPVIknock-out mice were also immunized with CGP6 and human platelets.

Immunogens except p-target were injected intraperitoneally. P-target wasinjected intradermally. Recombinant or purified proteins were injectedas an emulsion with adjuvant (Titermax Gold, Cytrx Corporation). Some ofthe antigens were immunized with mouse IL-6 (500 U/injection) to boostthe immune system. Animals were immunized until a serum titer between10-50,000 was obtained.

Monoclonal antibodies were produced by conventional hybridomatechnology. As a fusion partner, P3U1 cells were used. For screening forpositive hybridoma clones, FACS analysis was performed in the Epic AltraFACS analyzer by Beckman Coulter using GPVI-FcRγ expressing CHO cells.For CGP6-immunized animals, FACS analysis was performed with bothGPVI-FcRγ-transfected and non-transfected wild-type CHO cells todistinguish clones that produced non-GPVI antibodies e.g. antibodies toCHO cell-related antigens.

Hybridoma cells producing anti-GPVI antibodies were then grown in mediumcontaining 10% fetal calf serum (Invitrogen Corporation, Calif.) (whichcontained negligible amounts of bovine IgG <1 μg/ml serum). IgG waspurified from the cell-free culture supernatant by protein G-Sepharose(Amersham Biosciences, N.J.) or Protein A-Sepharose (AmershamBiosciences, N.J.) affinity chromatography on a Waters 650 system(Waters Corporation, Mass.). Mouse IgG was purified by affinitychromatography on Protein G-Sepharose. Hamster IgG was purified onProtein A-Sepharose. Protein G-bound IgG was eluted from the affinitymatrix with low pH glycine (pH 2.75), collected into basic solution toneutralize the acid, and dialyzed in saline for use in functionalassays. Protein—A bound hamster IgG was eluted with a pH gradient7.5-3.0. Antibody was eluted at pH 4.5. In most cases, antibody was >90%pure when analyzed in Agilent 2100 Bioanalyzer (Agilent Technology).

Bivalent F(ab′)₂ fragments were prepared from intact IgG by papaindigestion using standard methods. A solution of IgG (5 mg/ml) was madein 100 mM citric acid, pH 6.5 and 5.0 mM EDTA and digested for 15 hrs at37° C. with pre-activated papain (cysteine free) at an enzyme to IgGratio of 1:50 (wt/wt). The reaction was quenched with freshly preparediodoacetamide and F(ab′)₂ fragments were separated from undigested IgGand Fc by ion exchange chromatography on a MonoQ column (AmershamBiosciences, N.J.). Fractions containing F(ab′)₂ were pooled,concentrated and reduced/alkylated to obtain monovalent Fab fragmentsaccording to the method of Parham et al., J. Immunol. Meth. 53: 133-173(1982). Finally, Fab fragments were purified to homogeneity by sizeexclusion chromatography on Superdex 75 (Amersham Biosciences, N.J.).Direct conversion of IgG into monovalent Fabs by papain in the presenceof cysteine was avoided because in a few cases, papain over-digested theIgG, giving rise to unstable and smaller-sized Fab fragments.

Initially, wild-type Balb/c mice were immunized as described above withimmunogens including purified GPVI from human platelets (“nGP6”),plasmid containing GPVI cDNA (“p-target”), and CHO cells expressing theGPVI-FcRγ chain complex (“CGP6”). See Table 1. After screening more than8500 clones, 3 clones with significant but moderate affinity to GPVIwere identified (two from CGP6 and one from PGP6). Surprisingly, morethan 5500 clones arising from immunizations with p-target, nGPVI, andPAGP6 did not yield a single GPVI-positive clone with biologicalactivity. In an attempt to obtain antibodies with enhanced affinity andbiological activity, a different species of animals was immunized.Armenian hamsters were immunized with CGP6 because this immunogenproduced two positive clones in wild-type mice. Because human plateletsexpress native GPVI on their surface, washed human platelets were alsoused as immunogens. Seven GPVI-positive clones were obtained from theCGP6 immunizations and one clone from human platelet immunization. SeeTable 2.

TABLE 1 Immunization of Wild-type Mice Immunogen # of clones screened #of positive clones p-target 1192 0 CGP6 1953 2 PGP6 1064 1 nGP6 3797 0PAGP6  764 0 Total 8770 3

TABLE 2 Immunization of Hamsters Immunogen # of clones screened # ofpositive clones CGP6 1547 7 human platelets 1414 1 (OM3) Total 2961 8

GPVI knock-out (GPVI-KO) mice were then used as hosts for immunization.The GPVI-KO mice lack GPVI and do not respond to high doses of collagenand to convulxin (a GPVI specific agonist). Therefore, it was theorizedthat injected GPVI may be more antigenic in GPVI-deficient mice and maytherefore produce GPVI antibodies having high affinity for GPVI-peptide.The results of immunization of GPVI-KO mice are shown in Table 3.Immunization of GPVI-KO mice with washed human platelet suspensions didnot produce any positive clones. However, eight clones were obtainedfrom the immunization of GPVI-KO mice with CGP6, which had high affinityto GPVI as judged by the large rightward shift in fluorescence intensityin the FACS analysis.

TABLE 3 Immunization of GPVI Knock-Out Mice Immunogen # of clonesscreened # of positive clones CGP6 3889 8 (including OM1, OM2 and OM4)human platelets  397 0 Total 4286 8

IgG immunoglobulins were then obtained from the GPVI-positive clones: 3clones from wild type mice, 8 clones from hamster fusions, and eightclones from GPVI-KO mice fusions. IgG was purified by affinitychromatography using either Protein G (wild-type and GPVI-KO mice) orProtein A (hamsters), as described above. Purified antibodies from allclones induced full platelet aggregation at relatively smallconcentrations of IgG at 0.1-7 μg/ml (17 clones) and 10-30 μg/ml (2clones), suggesting that these antibodies cross-linked GPVI and FcR IIA,or cross-linked GPVI molecules. To exclude the possibility ofGPVI-FcRIIA cross linking, F(ab′)2 fragments were prepared. Inpreliminary studies, F(ab′)₂ fragments prepared from two antibodiesactivated human platelets, although several fold higher concentrationswere needed compared to intact IgGs. This confirmed that GPVI crosslinking by the bivalent antibodies may be the cause of the observedplatelet activation. In order to avoid GPVI cross linking, all F(ab′)₂fragments were converted to Fab fragments by reduction/alkylation.Resulting Fab fragments did not activate human platelets when tested upto several fold higher concentrations at which intact IgG activateshuman platelets.

EXAMPLE 2 GPVI Specific Antibodies are Potent Inhibitors ofCollagen-Induced Platelet Aggregation and Adhesion

The inhibitory potential of the Fab fragments prepared according toExample 1 was tested on collagen-induced platelet functions, includingcollagen-induced platelet aggregation and adhesion of platelets toimmobilized collagen under static and flow conditions.

The antibodies were tested for in vitro platelet aggregation as follows.A collagen dosing experiment was first performed to determine the amountof collagen that would give 70-90% platelet aggregation within 5 minutesof its addition because collagen response varies among individuals.Moreover, the type of collagen used in the assays can dramaticallyaffect the response. From experience, acid insoluble equine tendoncollagen (Nycomed, Germany) provided the greatest platelet aggregationresponse. Nieswandt (J. Biol. Chem. 275:23998-24002, 2000, and U.S.Patent Publication No. 2002/0141992), Lecut et al. (J. Thrombosis andHaemostasis 1:2653-2662, 2003), and Moroi et al. (Thromb. Haemost.89:996-1003, 2003) also used acid insoluble equine tendon collagen,whereas others used a less responsive form of collagen, for example,bovine collagen type I fibers (Smethurst et al., Blood 103:903-911,2004, and WO 03/054020). In the assays exemplified below, 1-4 μg/ml acidinsoluble equine tendon collagen (Nycomed, Germany) was used to examinethe inhibitory effect of a particular Fab preparation on plateletaggregation. The type of collagen used is same as that used in Qian etal. (Human Antibodies 11:97-105, 2002), WO 01/00810, WO 02/080968, andtheir related applications; but as discussed below, the GPVI antibodiesof the present invention exhibit significantly greater inhibitory effectcompared to the GPVI antibodies disclosed in Qian, WO 01/00810, and WO02/080968.

The platelet aggregation assay was performed by collecting blood in 1/10volume of 3.8% trisodium citrate as an anticoagulant (Nakamura et al.,J. Biol. Chem. 273(8):4338-44, 1998). Platelet rich plasma (PRP) wasobtained by centrifugation of whole blood at 180×g for 15-20 minutes atroom temperature. Platelets were counted and adjusted to 3-4×10⁸platelets/ml in platelet poor plasma prior to performing plateletaggregation studies. All experiments were performed within 3-4 hrs afterblood collection and PRP was maintained at room temperature the entiretime. Aggregation studies were performed in a four channel aggregometerAG10 (Kowa, Japan) which measures the kinetics of particle formation bylaser scattering and aggregation by light transmission at 650 nm in thevisible region of the light spectrum. PRP was incubated at 37° C. withvarying amounts of Fab fragments for 10 minutes (5 minutes withoutstirring followed by additional 5 minutes with stirring) prior to theaddition of acid insoluble equine tendon collagen (Nycomed, Germany) andaggregation was monitored for an additional 5-10 minutes.

Platelet adhesion to collagen under static conditions was examined usinga modified procedure of Nakamura et al., J. Biol. Chem. 273:4338-4334(1998) described earlier. Briefly, washed platelets were incubated withdesired amounts of Fab fragments for 30 minutes in the presence andabsence of Mg²⁺ at room temperature prior to their addition tocollagen-coated wells (2 μg/well with acid insoluble equine tendoncollagen) (Nycomed, Germany). After 60-90 minutes of incubation at roomtemperature, unadhered platelets were removed by gentle washing withbuffered saline and the adhesion was quantified by determining the LDHcontent of adhered platelets using a commercially available LDH kit(Promega, Mass.).

Platelet adhesion to immobilized collagen under flow conditions wasmeasured in a flow chamber developed by Glycotech (Rockville, Md.).Whole blood, anticoagulated with recombinant hirudin (50 units/ml), wasincubated with a solution containing Fab fragment for 15 minutes at 37°C. prior to being drawn through a collagen-coated (5 μg/cm² acidinsoluble equine tendon collagen) (Nycomed, Germany) flow chamber athigh shear (2600 sec⁻¹, 2 min). Unadhered platelets were washed withphosphate buffered saline and the adhered platelets were fixed withglutaraldehyde (0.5% w/v, 1 h) and stained with toludine blue/sodiumborate (0.05%, 5 min). Surface coverage by platelets was estimated bydigital image analysis. The average from 10 non-overlapping images wasused to determine percentage surface area coverage.

Of the antibodies selected, Fab fragments from four IgGs, OM1, OM2, OM3,and OM4, inhibited collagen-induced platelet aggregation on humanplatelets. See Tables 2, 3, and 4. The order of inhibitory potency ofthe Fab fragments against collagen-induced platelet aggregation wasOM1>OM2>OM3>OM4. Average IC₅₀ and SD values of each antibody forinhibition of collagen-induced platelet aggregation are shown in Table4.

Cross reactivity of each antibody to rat, dog, and monkey platelets werealso tested. Monkey and dog blood was purchased from Covance ResearchInc, Vienna, Va. Sprague Dowley rats were obtained from Charles RiverLaboratories, Willmington, Mass. All four Fab fragments inhibitedcollagen-induced aggregation of monkey platelets. Interestingly, OM4cross-reacted with rat platelets. These GPVI specific antibodies areuseful tools for testing the effect of GPVI specific antibodies inanimal models. Hybridomas producing OM1, OM2, OM3, and OM4 antibodieswere deposited with the American Type Culture Collection (Manassas, Va.)on Apr. 29, 2004, as ATCC Nos. PTA-5938, PTA-5939, PTA-5940, andPTA-5941, respectively.

TABLE 4 Effect of Anti-GPVI Fab Fragments on Collagen-InducedAggregation of Human Platelets and Cross-Reactivity to Non-HumanPlatelets Fab Fragment IC₅₀ (μg/ml)* Cross reactivity** ID Average SDRat Dog Monkey OM1 0.575 0.049 (−) (−) (+) OM2 1.69 0.506 (−) (−) (+)OM3 3.02 0.768 (−) (−) (+) OM4 7.0 5.5 (+) (−) (+) ReoPro ® 1.71 0.345(−) (+) (+) 7E3 F(ab′)₂ n/d*** n/d (+) n/d n/d *Dose response curve wereobtained using platelets from 3 different subjects. IC₅₀ values werecalculated by non-regression analysis. Values are average ± SD from 3experiments. **Cross reactivity with animal platelets was based on theability of individual Fab fragments to inhibit collagen-induced plateletaggregation and by positive rightward shift in FACS analysis. A (+) signindicates inhibition of collagen-induced aggregation by Fab fragmentsand positive rightward shift while a (−) sign indicates no reaction inboth tests. ***Not determined

For comparison, REOPRO® (Centocor, Inc.), a widely used human-mousechimeric anti-GPIIb-IIIa Fab fragment, and a F(ab′)₂ fragment of theanti-GPIIb-IIIb monoclonal antibody 7E3 (Coller and Scudder, Blood66:1456-1459, 1985), was tested on several of the same donors under thesame conditions used to test the GPVI specific antibodies. REOPRO®inhibited collagen-induced platelet aggregation at an IC₅₀ of 1.71μg/ml. Thus, OM1 possessed greater inhibitory potential than REOPRO®,while OM2 was equipotent to REOPRO®. REOPRO® was 2-4 times more potentthan OM3 and OM4 in this assay. 7E3 F(ab′)₂ cross-reacted with ratplatelets, whereas REOPRO® did not (see Table 4).

The effect of Fab fragments on platelet adhesion under static conditionswas also tested. Adhesion was carried out in the presence (GPIa-IIa andGPVI-dependent) and absence (GPVI-dependent) of Mg²⁺. Mg²⁺-independentadhesion is solely dependent on the presence of GPVI and was inhibitedby all four Fabs at relatively low concentrations (IC₅₀ ranging from0.1-1 μg/ml) as shown in FIG. 2. OM1, OM2, and OM3 had similarinhibitory activity (IC₅₀ range 0.1-0.2 μg Fabs/ml) while OM4 required aslightly higher dose to achieve similar inhibition (IC₅₀ range 0.2-1 μgFabs/ml). The Mg²⁺-dependent adhesion process required relatively higherdoses of Fab fragments than those required for the Mg²⁺-independentadhesion process (see FIG. 2). However, none of the Fabs were able tocompletely block the Mg²⁺-dependent adhesion of platelets to collagen.

The effect of Fab fragments on Mg²⁺-independent platelet adhesion understatic conditions was repeated using even lower doses (0.001-1 μgFabs/ml). As shown in FIG. 3, OM2 and OM4 Fab fragments inhibitedplatelet adhesion by 40-60% at 0.1 μg Fab/ml and OM1 and OM3 Fabfragments inhibited adhesion by 10-20% at the same concentration. Thisdiscrepancy may be due to batch variation and donor variability. Inconclusion, the OM series of Fab fragments effectively inhibitGPVI-dependent (Mg²⁺-independent adhesion) at concentrations lower than0.5 μg/ml.

The inhibitory effect of anti-GPVI Fab fragments on platelet adhesionwas also tested under conditions closer to in vivo situations, asdescribed above. Fab fragments of OM1, OM2, OM3, OM4 significantlyinhibited platelet adhesion to immobilized collagen under high shearconditions (2600 sec⁻¹). Compared to a control sample, anti-GPVI Fabsinduced dramatic changes in the size and morphology of the aggregates(FIG. 4). In comparison, REOPRO® (Centocor, Inc.) also prevented theformation of aggregates but a uniform layer of single platelets wasobserved on collagen fibers (FIG. 4).

The morphology of the aggregates is a result of two events: (1) the areacovered by primary monolayer of platelets and (2) subsequent formationof aggregates thus adding a volume dimension to the over all picture.The dramatic reduction in aggregate formation along with surfacecoverage by the anti-GPVI Fab suggests that GPVI is not only involved inthe primary adhesion process but that it also plays an important role inpost-adhesion events, including platelet activation and subsequentthrombus growth.

Aggregation and adhesion assays demonstrate that the Fab fragments ofGPVI specific antibodies of the invention, such as OM1, OM2, OM3, andOM4, are potent inhibitors of platelet functions. They are more potentat inhibiting collagen-induced platelet aggregation than the Fabfragment of the mouse monoclonal antibody 9O12.2 generated from miceimmunized with a cDNA encoding recombinant soluble GPVI-Fc (rsGPVI-Fc)fusion protein as described in Lucet et al. (J. Thrombosis andHaemostasis 1:2653-2662, 2003), WO 02/80968, and US Patent Publication2004/0253236. Additionally, the OM antibodies are more potent atinhibiting Mg²⁺-independent adhesion to collagen than the 9012.2 Fabfragment.

Nieswandt (J. Biol. Chem. 275:23998-24002, 2000, and U.S. PatentPublication No. 2002/0141992) reported a rat monoclonal antibody tomouse GPVI, JAQ1. However, saturating concentrations of JAQ1 (20 μg/ml)only displayed a limited inhibitory effect on collagen-induced plateletaggregation (see U.S. Patent Publication 2002/0141992, paragraph 29).Furthermore, JAQ1 did not recognize human GPVI in FACS analysis orWestern blotting in our hands or in others (see Takayama et al., J.Thrombosis and Hemostasis 14: 75-81, 2003).

Others have generated single chain Fvs (ScFvs). For example, Qian et al.(Human Antibodies 11:97-105, 2002) reported a single chain Fv (ScFv)antibody of GPVI that had an IC₅₀ of 80-90 μg/ml in a collagen-inducedplatelet aggregation study using 2 μg/ml of the same collagen used inthe present invention. Thus, the GPVI specific antibodies of theinvention are significantly more potent in inhibiting collagen-inducedplatelet aggregation compared with Qian et al. ScFvs also reported in WO01/00810, WO 02/80968, and their related applications required asignificantly greater concentration (110-150 μg/ml of ScFv) forinhibiting collagen-induced platelet aggregation compared with the GPVIspecific antibodies of the present invention.

Similarly, Smethurst et al. (Blood 103:903-911, 2004, and WO 03/054020),reported a ScFv antibody of GPVI that had an IC₅₀ of 12-16 μg/ml incollagen-induced platelet aggregation. In comparison, OM1, OM2, OM3 andOM4 are more potent than Smethurt's ScFv. Additionally, although U.S.Pat. Nos. 6,245,527 and 6,383,779 disclose anti-GPVI antibodies, they donot provide any examples of anti-GPVI antibodies that are as potent atinhibiting collagen-induced platelet aggregation as those of the presentinvention.

EXAMPLE 3 GPVI Specific Antibodies Inhibit Collagen-Induced Secretionand Thromboxane A₂ Formation

Fab fragments of the GPVI specific antibodies of the invention were alsotested for their effect on collagen-induced secretion and thromboxane A₂(TXA₂) formation. Secretion refers to agonist-induced release ofbioactive contents from alpha and dense granules from platelets.

One way to quantify agonist-induced release is to measure ATP content inthe medium by luciferase assay using chemiluminescence method.Platelet-rich plasma (PRP) was tested for collagen-induced ATP secretionusing a Lumi-aggregometer (Chronolog Corporation, PA) and aluciferase-luciferin reagent. The Lumi-aggregometer simultaneouslymeasures the agonist-induced platelet aggregation and ATP secretion.Briefly, human blood was drawn directly into 3.8% trisodium citrate witha syringe (9:1 volume blood:citrate). Platelet-rich plasma (PRP) wasprepared by centrifugation at 180×g for 15 minutes. PRP (360 μl) wasmixed with 40 μl luciferase-luciferin reagent (Chrono-lume; ChronologCorporation, PA) and the mixture was incubated with varying amounts oftest Fabs, REOPRO®, or control for 5 minutes at 37° C. 1-4 μg/ml ofcollagen (acid insoluble equine tendon collagen) (Nycomed, Germany) wasadded at five minutes and aggregation and ATP secretion were monitoredfor eight minutes. At the end of the reaction (10-11 minutes), a knownamount of ATP solution was added to obtain a deflection which was usedto calculate the ATP amount secreted by platelets upon agonistchallenge.

Thromboxane A₂ formation were measured in parallel samples used above.Ten minutes after the collagen addition step above, 500 μL of stopsolution (50 mM EDTA, 2 mM Indomethacin in 130 mM NaCl) was added to 200μL of PRP to terminate thromboxane A₂ formation. The suspension wascentrifuged at 1,000×g for 10 min at 4° C. The supernatant was saved andfrozen at −20° C. until tested for thromboxane B₂, which is a stablemetabolite of thromboxane A₂. Thromboxane B₂ was quantified by using acommercially available kit (Thromboxane B₂ Biotrak Assay, Amersham).

All Fab fragments of OM1, OM2, OM3, and OM4 antibodies potentlyinhibited collagen-induced ATP release from human platelets (Table 5).OM1 showed inhibition of more than 90% at 1 μg/mL. OM2, OM3, and OM4also attained inhibition of more than 90% at 3 μg/mL. REOPRO® was lesseffective at inhibiting ATP secretion than the anti-GPVI antibodies,suggesting that anti-GPVI Fabs are better inhibitors of collagen-inducedATP release. Secondary agonists released from platelets are known tosynergize thrombus growth. Therefore, inhibitors of collagen-inducedsecretion, such as the GPVI specific antibodies of the invention, arepotent inhibitors of thrombus growth.

TABLE 5 Effect of Anti-GPVI Fab Fragments on Collagen-Induced ATPRelease From Human Platelets* concentration % inhibition antibody(μg/mL) (mean ± SD) OM1 1 97.6 ± 2.1 3 96.8 ± 6.3 OM2 1  63.6 ± 41.6 397.7 ± 2.7 OM3 1  72.8 ± 23.7 3 94.2 ± 0.4 OM4 1  50.0 ± 29.5 3 91.3 ±5.2 ReoPro ® 1  18.0 ± 13.5 3  56.9 ± 31.7 *Results were obtained from 4different platelet donors.

Collagen-induced thromboxane A₂ generation was also strongly inhibitedby Fab fragments of OM1, OM2, OM3, and OM4 (Table 6). Among the four Fabfragments tested, OM1 showed inhibition of more than 90% at 1 μg/mL. OM2and OM3 also attained inhibition of more than 90% at 3 μg/mL. OM4inhibited thromboxane A₂ formation by about 86% at 3 μg/mL. In contrast,REOPRO® showed little or no inhibitory effect on thromboxane A₂formation at 1 and 3 μg/mL. It has also been shown that blockade of GPVIinhibits the generation of TXA₂ and expression of an activated IIb-IIIacomplex on collagen-adhered platelets (Nakamura et al., J. Biol. Chem.273:4338, 1998; Nakamura et al., J. Biol. Chem. 274:11879, 1999).Therefore, inhibition of TXA₂ generation by anti-GPVI Fab fragmentssuggests that the GPVI specific antibodies of the invention inhibit theupstream signalling events leading to the expression of activatedIIb-IIIa complex and ultimately, followed by an attenuation in thrombusgrowth.

TABLE 6 Effects of Fab Fragments of Anti-GPVI Antibodies onCollagen-Induced Thromboxane A₂ Formation in Human Platelets*concentration ng/3 × 10⁸ platelets antibody (μg/mL) (mean ± SD) control104.3 ± 37.4  OM1 1 5.0 ± 3.4 3 2.9 ± 2.7 OM2 1 31.5 ± 36.5 3 5.0 ± 3.3OM3 1 24.1 ± 10.6 3 6.9 ± 4.8 OM4 1 28.7 ± 8.1  3 14.5 ± 4.9  ReoPro ® 177.8 ± 35.1 3 106.7 ± 70.9  *Results were obtained using platelets from4 different donors

EXAMPLE 4 Binding Affinities and Reactivity of Fab Fragments ofAnti-GPVI Antibodies to GPVI

Binding affinities were determined according to the method of Fujimuraet al. Thromb Haemost 87:728-34 (2002). ¹²⁵I-labeled antibodies used inthe determination of binding affinities were prepared from unlabeledIgGs according to the Iodo-beads method (Pierce, Ill.). Briefly, oneIodo-bead was soaked for 5 minutes in iodination buffer containing 0.5mCi carrier-free Na¹²⁵I (Amersham) followed by the addition of acandidate IgG (100 μg). After 5 minutes of incubation at roomtemperature, the reaction mixture was applied to a PD10 column(Amersham) to separate ¹²⁵I-bound IgG from free iodine. Fractionscontaining ¹²⁵I-IgG were eluted from the column and a small volume (1μl) was subjected to TCA precipitation. Both the precipitated pelletsand the resulting supernatants were counted in a gamma counter toquantify the incorporation of ¹²⁵I in IgG. Fractions with maximal countsin the precipitate (<95%) were pooled and read in a spectrophotometer at280 nm to determine protein concentration. A known volume was recountedin a gamma counter to obtain specific activity of the labeled IgG. Thespecific activity among various antibodies ranged from 0.33-0.97 μCi/ugIgG.

Binding affinities were determined by incubation of washed humanplatelets (5×10⁸/ml) with 1 nM ¹²⁵I-IgG in the presence and absence ofvarious concentrations of unlabelled homologous IgG (0-500 nM) for 1 hr.The free and bound radioactivities were separated by layering themixture over BSA (10% solution in saline) solution and centrifuged at15, 000×g for 10 minutes. After careful removal of both the supernatantand the BSA cushion, the tip of the tube was cut and radioactivity ofthe pellet was counted in a gamma counter. Eight to ten triplicate-pointcompetition binding isotherms were developed for evaluating the bindingof individual IgG. The data was analyzed using the non-linear regressionanalysis software, Prism (GraphPad Software Inc. CA).

Binding affinity experiments revealed that all four antibodies boundavidly to platelets with affinities ranging from 0.7-1.7 nM (Table 7).The antibodies of the invention were compared with the bindingaffinities of antibodies reported in the art: REOPRO®(Kd=6.25±2.6×10⁻⁹M) (from Sassoli et al., Thromb Haemost 85:868-902,2001), scFv-10B12 (Kd=7.9×10⁻⁷M) (from Smethurst et al., Blood103:903-91, 2004), and 9012.2-IgG (Kd=18×10⁻⁹M) (from WO 02/080968).REOPRO® and scFv-10B12 are monovalent fragments whereas the OM seriesused in this example and 9O12.2 are IgGs. All four OM antibodies boundwith higher affinity to human platelets than reported for REOPRO®,scFv-10B12, and 9012.2-IgG (Table 7). Monovalent fragments generallyhave somewhat reduced affinities compared with their correspondingintact IgG; however, that reduction would not be expected to besignificant.

TABLE 7 Binding Affinities of Anti-GPVI Antibodies Antibody ID Kd μgIgG/ml OM1  1.724 ± 0.22 × 10⁻⁹M  0.258 ± 0.033 OM2  0.723 ± 0.093 ×10⁻⁹M  0.108 ± 0.0103 OM3  0.8187 ± 0.14 × 10⁻⁹M  0.1220 ± 022    OM4 0.785 ± 0.25 × 10⁻⁹M  0.117 ± 0.037 ReoPro ®*   6.25 ± 2.6 × 10⁻⁹M  0.31 ± 0.13 scFv - 10B12** 7.9 × 10⁻⁷M  23.7 9O12.2 - IgG*** 18 × 10⁻⁹M 2.7 The binding study of the OM series is from a single experimentusing platelets from different individuals. Each data point was run intriplicate. *Data from Sassoli et al. Thromb Haemost 85: 868-902 (2001).**Data from Smethurst et al., Blood 103: 903-911 (2004). 10B12 is ahuman specific scFv antibody against GPVI obtained from a phage displaymethod. ***Data from WO 02/080968. 9O12.2 is a monoclonal anti-GPVIantibody.

Western blotting analysis was performed to determine the reactivity ofthe OM series antibodies to GPVI from various species. Platelets(1×10⁸/ml) from various species were solubilized in 2% SDS containingEDTA, EGTA, PMSF and NEM (1 mM each). Proteins were separated on 4-20%pre-cast Tris-glycine gradient mini gels (Invitrogen, Carlsbad, Calif.),and the resolved proteins were transferred to nitrocellulose membranes(Invitrogen, Carlsbad Calif.). Individual lanes were cut and blockedwith 5% skim milk in TBS-T (10 mM Tris.HCl pH 7.4, 150 mM NaCl, and 0.5%Tween 20) for 60 minutes. Nitrocellulose strips were incubated with OMantibodies (2 μg/ml) or biotinylated convulxin overnight at 4° C.Membranes were washed extensively with TBS-T and probed with eitherHRP-conjugated goat anti-mouse IgG (OM1, OM2, and OM4), HRP-conjugatedgoat anti-hamster IgG (OM3) or streptavidin-HRP (biotinylated convulxin)for 1 hr at room temperature. Membranes were washed three times with alarge excess of TBS-T. Immune reactive bands were visualized using anenhanced chemiluminescence detection system (ECL-Amersham PharmaciaBiotech, Little Chalfont, UK).

All four OM antibodies reacted with denatured GPVI from human plateletson immunoblots (FIG. 5). Similar reactivity was seen with monkeyplatelets (blots not shown). None of the antibodies reacted with mouse,pig, dog, rabbit or guinea pig platelets. Only OM4 reacted positivelywith rat platelet lysate. These data suggest that all of the OM seriesantibodies recognize GPVI in platelets from humans and monkeys while OM4additionally recognizes GPVI in rat platelets.

EXAMPLE 5 Complementarity Determining Regions (CDRS)

The sequences of the complementarity determining regions (CDRs) of OM1,OM2, OM3 and OM4 were determined.

Total RNA was isolated from OM1, OM2, OM3, and OM4 hybridomas usingTRIzol (Invitrogen). cDNA was synthesized with the SuperScriptFirst-Strand System (Invitrogen) using random primers. DNA sequencescorresponding to the variable regions of antibodies were then amplifiedby polymerase chain reaction using Platinum Pfx DNA Polymerase(Invitrogen) with Heavy Primers or Light Primers mix (Amersham). Theamplified DNA was ligated into pCR4-TOPO vector (Invitrogen) and theresulting construct was transformed into chemically competent cellsusing the TOPO TA Cloning kit (Invitrogen). Transformed cells werecultured in the presence of kanamycin and the amplified plasmid wasisolated using the E.Z.N.A. Plasmid Miniprep Kit II (Omega Bio-tek).Sequencing of inserted DNA was performed on the ABI PRISM 310 GeneticAnalyser using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit(Applied Biosystems). DNA sequences were analyzed and converted intoamino acid sequences using the OMIGA 2.0 software (Oxford Molecular).

The CDRs of OM1, OM2, OM3, and OM4 are shown in Table 8.

TABLE 8 CDR Sequences of Anti-GPVI Antibodies OM1 H1: SYWMN (SEQ IDNO:1) H2: MIHPSDSETTLNQKFKD (SEQ ID NO:2) H3: DDYYDSSSHALDY (SEQ IDNO:3) L1: RASQSVSTSTYSYIY (SEQ ID NO:4) L2: FASYLES (SEQ ID NO:5) L3:QHIWEIPWTF (SEQ ID NO:6) OM2 H1: DHYIS (SEQ ID NO:7) H2:WIYPGYGNIRYNEKFKG (SEQ ID NO:8) H3: SADGYFRYFDV (SEQ ID NO:9) L1:RASGNIHNYLA (SEQ ID NO:10) L2: NSEILAD (SEQ ID NO:11) L3: QHFWTAPFTF(SEQ ID NO:12) OM3 H1: DFYMN (SEQ ID NO:13) H2: SISGGSSDIKYADVVKG (SEQID NO:14) H3: WGDHWDLDY (SEQ ID NO:15) L1: QASQNIGNELN (SEQ ID NO:16)L2: GASSLYP (SEQ ID NO:17) L3: KQDLNYPITF (SEQ ID NO:18) OM4 H1: SFGMH(SEQ ID NO:19) H2: FISSGSSTIYYADIVKG (SEQ ID NO:20) H3: SGYANAMDY (SEQID NO:21) L1: KASQDVSPAVT (SEQ ID NO:22) L2: WASTRHT (SEQ ID NO:23) L3:QQHYSFPWTF (SEQ ID NO:24)

EXAMPLE 6 GPVI and Thrombosis

The role of GPVI in thrombosis had been shown in a prior study usingGPVI-depleted or FcRγ-KO/GPVI deficient mice (Nieswandt et al., The EMBOJournal 20:2120-2130 (2001)). Both the GPVI-depleted or FcRγ-KO/GPVIdeficient mice lack the FcRγ chain. In this example, the involvement ofGPVI in thrombosis was confirmed using GPVI-KO mice that do not lack theFcRγ chain.

GPVI knock-out mice were developed as described above. Platelets fromthese mice failed to respond to high dose collagen (20 μg/ml) and to theGPVI-specific agonist, convulxin (3 μg/ml), but responded normally toADP (5 μM) (FIG. 6). Heterozygous GPVI-deficient mice which produce halfthe amount of GPVI compared with wild-type showed reduced responses tocollagen and convulxin but normal responses were observed upon increasein agonist dose (FIG. 6). These observations confirm the role of GPVI asa dominant collagen receptor on the platelet surface.

The role of GPVI in thrombosis and homeostasis was first tested byco-injection of collagen-epinephrine, which normally induces a lethalpulmonary thrombo-embolism (see Table 9). Mice were anaesthetized withketamine/xylazine (150/15 mg/kg, IP) and a mixture of collagen andepinephrine (800/60 μg/kg) was injected into the right jugular vein. Theanimals were then observed for 15 minutes and categorized as follows:(a) animals that succumbed to death within 10 minutes of the injection,and (b) animals that survived showed transient respiratory distress,which alleviated within 10 minutes. The surgical wounds of survivinganimals were sutured and the animals were returned to the animalfacility. Approximately 83% (15 out of 18) of GPVI wild-type mice diedwithin 5 minutes of injection. All heterozygous GPVI-deficient mice (18out of 18) died within five minutes of injection. In contrast towild-type and heterozygous animals, approximately 55% of the GPVI-KOmice (homozygous) survived the lethal injection of collagen andepinephrine, suggesting that GPVI plays an important role in pulmonarythromboembolism induced by the injection of collagen.

TABLE 9 The Role of GPVI in Pulmonary Thromboembolism number of animalsGenotype survived % Survival Wild-type (+/+) 3/18 ~16 Heterozygous (+/−)0/18 0 Homozygous (−/−) 10/18  ~55

As shown in Table 10, GPVI knock-out mice had essentially same tailbleeding time as wild-type and heterozygous mice. Bleeding times of theGPVI knock-out mice were compared to those of knock-out mice deficientin β3 integrin, which is also found on platelets and is involved inplatelet aggregation and thrombosis (Kairbaan et al., J. Clin. Invest.103:229-238, 1999). In contrast to the GPVI-deficient mice, thehomozygous β3 knock-out mice bled continuously. Therefore,administration of anti-GPVI Fab fragments in vivo may be safer as theyshould not significantly affect bleeding time because the bleeding timein GPVI-knockout mice is similar to that of wild-type mice.

TABLE 10 Tail Bleeding Time in Wild-Type, Heterozygous and HomozygousMice: Comparison With β3 Knock-Out Mice Tail bleeding time (seconds)Genotype GPVI-knock out β3-knock out* Wild-type (+/+) 191 ± 186 (n = 9) 156 ± 89 (n = 15) Heterozygous (+/−) 190 ± 193 (n = 14) 156 ± 99 (n =15) Homozygous (−/−) 165 ± 110 (n = 13) >600 (n = 15)** *J Clin Invest103: 229, 1999 **Significantly different from wild-type and heterozygousmice. In most cases, bleeding had to be stopped manually to preventdeath

To further confirm the role of GPVI in thrombosis, whole blood fromwild-type, heterozygous and GPVI-KO mice were perfused onto type Icollagen-coated cover slips at a shear stress of 2600 sec-1. Plateletsfrom GPVI-KO mice failed to adhere to collagen fibers while plateletsfrom wild-type and GPVI-heterozygous mice adhered to collagen fibers andformed large thrombi. There was no difference in surface coverage andthrombus morphology between wild-type and GPVI-heterozygous mice (FIG.7).

EXAMPLE 7 Effect of OM2 Fab Fragment on Ex Vivo Collagen-InducedPlatelet Aggregation and Skin Bleeding Time in Cynomolgus Monkeys

Dose-escalation study. The OM2 Fab fragment was further evaluatedbecause it showed the strongest inhibitory effect among the OMantibodies in an in vitro collagen-induced platelet aggregation assayusing platelets from Cynomolgus monkeys. The OM2 Fab fragment wasadministered by intravenous injection to Cynomolgus monkeys inescalating doses and its effect on ex vivo collagen-induced plateletaggregation and skin bleeding time were evaluated. REOPRO® was tested ina similar manner.

Under inhalation anesthesia, the OM2 Fab fragment or REOPRO® wasinjected intravenously into the cephalic vein of the forearm at 1 hrintervals. Thirty minutes after each injection, blood was collected forthe measurement of collagen-induced platelet aggregation. Platelet richplasma (PRP) and platelet poor plasma (PPP) were prepared by sequentialcentrifugation of blood that was anti-coagulated with trisodium citrate.Platelet aggregation was measured by the turbidimetric method 1 hr afterblood collection. Concentrations of collagen used to induce plateletaggregation were between 5 and 20 μg/mL, depending on the responsivenessof the platelets from each monkey. Skin bleeding time was measured 30min after each injection of the antibody by compressing the muscle ofthe forearm with a manchette at 40 mmHg and incising the skin with aTriplett Bleeding time device (Helena Laboratory). Blood flowing fromthe wound was absorbed with filter paper and bleeding time was measureduntil bleeding stopped, or at 1800 sec. Cumulative dose of antibody wasused to evaluate the effects of the antibodies.

The OM2 Fab fragment exerted a dose-dependent inhibitory effect oncollagen-induced platelet aggregation (FIG. 8). At the cumulative doseof 0.2 mg/kg or higher, the OM2 Fab fragment inhibited plateletaggregation by more than 80%.

REOPRO® also exerted a dose-dependent inhibitory effect oncollagen-induced platelet aggregation (FIG. 8) but inhibited plateletaggregation by more than 80% at a cumulative dose of 0.35 mg/kg orhigher.

OM2 Fab fragment prolonged skin bleeding time only slightly (2.4 timesthe baseline value) at a cumulative dose of 0.8 mg/kg (FIG. 9). Althoughthe OM2 Fab fragment slightly prolonged bleeding time at a cumulativedose of 18.8 mg/kg, bleeding time did not exceed the bleeding timeobserved at 0.8 mg/kg.

In contrast, REOPRO® prolonged skin bleeding time dramatically (FIG. 9).At a cumulative dose of 0.35 mg/kg, the average bleeding time was 5times longer than the baseline value. Additionally, the prolongation ofbleeding time by REOPRO® was dose-dependent. At a cumulative dose of1.55 mg/kg, bleeding time was 9.5 times longer than baseline level.

In summary, the OM2 Fab fragment showed an equally potent inhibitoryeffect on ex vivo collagen-induced platelet aggregation as exhibited byREOPRO®. However, the effect of OM2 Fab fragment on skin bleeding timewas much milder than that of REOPRO®. These results suggest thatblockade of GPVI has a superior risk/benefit ratio when compared to thatof GPIIb/IIIa blockade and therefore may be better suited for clinicaltreatment. Moreover, a red spot was observed at the injection site inone monkey after administration of REOPRO® at 0.4 and 0.8 mg/kg.Although this spot disappeared after several days, no abnormalities wereobserved after OM2 Fab administration.

Pharmacodynamics study. The change in the effects of the OM2 Fabfragment over time on ex vivo collagen-induced platelet aggregation andskin bleeding time was evaluated in three Cynomolgus monkeys after bolusintravenous injection. The change in effects by REOPRO® was similarlyevaluated.

The dose of 0.4 mg/kg was selected for this study because the OM2 Fabfragment and REOPRO® inhibited ex vivo collagen-induced plateletaggregation at 0.2 mg/kg and 0.35 mg/kg, respectively. After bolusintravenous injection of antibody into the cephalic vein of the forearm,blood was collected at 1, 2, 3, 6 and 24 hrs. Platelet aggregation wasmeasured 1 hr after each blood collection as described above under thedose-escalation study. Concentrations of collagen used to induceplatelet aggregation were 5 or 10 μg/mL in this study, depending on theresponsiveness of the platelets from each monkey. Bleeding time was alsomeasured at 1, 2, 3, 6 and 24 hrs after antibody administration asdescribed under the dose-escalation study.

The OM2 Fab fragment injected at 0.4 mg/kg inhibited ex vivocollagen-induced platelet aggregation by more than 80% at 1, 2, 3 and 6hrs after administration (FIG. 10). At 24 hrs after injection, plateletaggregation recovered nearly to basal level.

Similarly, REOPRO® injected at 0.4 mg/kg inhibited ex vivocollagen-induced platelet aggregation by more than 80% at 1 and 2 hrsafter administration (FIG. 10). However, in contrast to the OM2 Fabfragment, platelet aggregation recovered in a time-dependent manner: 73%inhibition at 3 hr, 47% inhibition at 6 hr and 6% inhibition at 24 hrafter administration.

As shown in FIG. 11, the OM2 Fab fragment slightly prolonged skinbleeding time between 1 and 6 hrs after administration (1.7 to 2.0 timeslonger than baseline level). Bleeding time returned to nearly baselinelevel at 24 hrs after injection, coincident with the recovery ofplatelet aggregation.

In contrast, REOPRO® significantly prolonged bleeding time at 1 hr afteradministration (10.7 times longer than baseline level) (FIG. 11).Prolongation of bleeding time became less prominent in a time-dependentmanner.

These results showed that the inhibitory half-life of the OM2 Fabfragment on platelet aggregation is longer than that of REOPRO®. Inaddition, these results again suggest that the risk/benefit ratio of theOM2 Fab fragment is superior to that of REOPRO®.

EXAMPLE 8 Effect of OM4 Fab Fragment on Ex Vivo Collagen-InducedPlatelet Aggregation, Bleeding Times, and Platelet Count in Rats

The OM4 Fab fragment was further tested for ex vivo collagen-inducedplatelet aggregation, tail and nail bleeding time in rats. Forcomparison, the 7E3 F(ab′)₂ fragment derived from an established murineantibody raised against human platelet glycoprotein complex GPIIb/IIIa(Collar et al. J. Clin Invest. 72:325-338, 1983) was tested in a similarmanner. 7E3 IgG was obtained from large cultures using a 7E3 hybridomaobtained from the ATTC. F(ab′)₂ fragments were prepared as described inCollar et al J. Clin Invest. 72:325-338, 1983. In preliminary studiesusing rats, the optimal dose of OM4 Fab and 7E3 F(ab′)₂ were determinedto be 20 and 10 mg/kg, respectively. The ex vivo collagen-inducedplatelet aggregation remained inhibited by OM4 Fab for 30 minutes afterwhich the inhibition reversed at 60-90 minutes, suggesting a fastclearance of OM4 Fab in rats. All observations were made at 20 minutesafter the administration of vehicle, test and reference antibody.

Adult male Sprague-Dawley rats were anesthetized with ketamine/xylazine.Heparin-filled catheters were inserted into the femoral vein, femoralartery, and carotid artery for drug administration, blood pressure/heartrate recording, and blood sampling, respectively. After theequilibration period, a small sample of blood (˜1.2 mL, anticoagulatedwith 10 U/mL heparin) was withdrawn from the carotid artery fordetermination of platelet count. The degree of platelet aggregationelicited by 1 μg/mL collagen was measured using a whole bloodaggregometer (Chrono-log, Havertown, Pa.). Nail-bleeding time was alsodetermined at this time by cutting one of the hind limb toenails at apoint that transected the nail pulp and by absorbing the blood every 15sec onto a piece of filter paper, without touching the cut surface ofthe nail. Nail-bleeding time was defined as the time elapsed betweencutting the nail and the point at which no further blood absorbed ontothe filter paper.

Vehicle, OM4 Fab, or 7E3 F(ab′)₂ composition was administered into thefemoral vein. At 20 minutes, a second blood sample was withdrawn foraggregometry and platelet count determination, as described above.Immediately following withdrawal of the final sample, nail and tailbleeding times were determined. Tail-bleeding time was determined byremoving the terminal 3 mm of the tail using a sharp scalpel blade andimmersing the distal 2-3 cm tail into 37° C. saline. Tail-bleeding timewas defined as the time elapsed between cutting the tail and the pointat which no more blood drew from the cut surface of the tail. Six ratswere used for each group (vehicle, OM4 Fab and 7E3 F(ab′)₂ fragment).

Both OM4 Fab and 7E3 F(ab′)₂ fragments produced a statisticallysignificant degree of inhibition of platelet aggregation at 20 min afterthe administration of the antibody fragments, in relation to the vehicle(P<0.05) (FIG. 12). The inhibition was highly reproducible, although thevariability was slightly greater in the OM4 Fab group. Blood pressureand body core temperature remained unchanged by the intravenousadministration of vehicle, OM4 Fab, and 7E3 F(ab′)₂ fragment compounds.

Nail bleeding time was dramatically prolonged by the administration ofthe 7E3 F(ab′)₂ fragment while the administration of OM4 Fab had noeffect in four out of six animals tested (FIG. 13A). In two animals, thenail bleeding time was prolonged to 26 and 31 minutes by theadministration of OM4 Fab. In contrast, the 7E3 F(ab′)₂ fragmentprolonged nail bleeding time beyond 30 minutes in all cases except onein which nail bleeding halted at 18 minutes (FIG. 13A). The mean nailbleeding time in animals receiving 7E3 F(ab′)₂ fragment wassignificantly greater than OM4 Fab and vehicle groups, as indicated bythe Student's t-test. OM4 Fab did not induce any significantprolongation of nail bleeding time when compared to vehicle.

Similar to the nail bleeding time, tail bleeding time was dramaticallyprolonged in all animals by the administration of 7E3 F(ab′)₂ fragmentwhile administration of OM4 Fab had no effect in four out of six animalstested (FIG. 13B). In two animals, the tail bleeding time was prolongedto 26 and >30 minutes. In contrast, the 7E3 F(ab′)₂ fragment prolongedtail bleeding time to beyond 30 minutes in all cases (FIG. 13B). Therewas no significant effect of intravenous administration of vehicle, OM4Fab and 7E3 F(ab′)₂ fragment on platelet count (FIG. 14).

The data obtained from this study clearly shows that OM4 Fab (20 mg/kg)and 7E3 F(ab′)₂ fragment (10 mg/kg) elicit similar degrees of inhibitionof collagen-induced platelet aggregation but 7E3 F(ab′)₂ significantlyprolonged bleeding time. OM4 Fab's ability to inhibit platelet functionwithout significantly affecting bleeding time suggests that it would betherapeutically beneficial. OM4 Fabs may provide similar positivebenefits as currently available platelet inhibitors without theirnegative bleeding side effects.

EXAMPLE 9 Effect of OM4 Fab Fragment in a Rat Arterial Thrombosis Model

The effect of the OM4 Fab fragment was also studied in an in vivoarterial thrombosis model in rats. Although various models of in vivothrombosis have been reported in the literature, a model developed byFolts (see e.g., Circulation 83(6 Suppl):IV 3-14, 1991) has been widelyused to test the efficacy of antithrombotic agents. This original modelwas developed in a canine coronary artery but for this study, the modelwas modified for testing on rat carotid artery. Briefly, the carotidartery was mechanically injured, followed by stenosis. Combination ofvascular injury and narrowing of the blood vessel (two conditionsmimicking the pathogenesis of thrombosis, i.e. arteriosclerosis andstenosis) results in the formation of a thrombus. The thrombus can thenbe mechanically dislodged and reformed by removal and replacement of thevascular occluder, respectively. This leads to cyclic flow reduction(CFR) as discussed in more detail below. Antithrombotic agents mayreduce the number of CFR or completely prevent the formation of CFR.

Rats were anesthetized with pentobarbital (50 mg/kg, i.p.) andmechanically ventilated with an intubation of the tracheal. A segment ofthe femoral vein was dissected and used for drug injection. The carotidartery was exposed via a midline incision in the ventral cervical areaand dissected free of connective tissue. A small flow probe (Transonic,1RB, Transonic Systems Inc., Ithaca, N.Y.) was placed distally on theartery to measure blood flow. Thrombosis was induced by applying twoconditions. First, an injury was induced by three consecutivecross-clamps of the middle exposed segment of the artery for 10 secondseach, with a needle holder having one ratchet click closed. Second, a50% reduction of the baseline blood flow was applied by inflating avascular occluder (1.5 mm inner diameter, In Vivo Metric, Healdsburg,Calif.), which consists of a C-shaped balloon that was placed on thesite of the injury. Blood flow was gradually reduced to zero withinabout 2-5 minutes due to the formation of a thrombus. The thrombus wasphysically dislodged by deflating the balloon, and blood flow wasimmediately restored. One CFR is counted when the blood flow reduces tozero after the 50% blood flow reduction and when blood flow is restored.After re-applying the 50% flow reduction, blood flow again graduallydecreased, resulting in the next CFR as a new thrombus formed. Thenumber of CFRs was counted during a 30 min observation period. Thenumber was also rounded to a half cycle.

In the pre-injury groups, either saline or OM4 Fab fragment at 20 mg/kgwas given by an intravenous bolus injection 2 min before mechanicalinjury was applied. CFRs were then recorded for 30 min.

In the post-injury groups, CFRs were initiated by endothelial injury and50% reduction of flow. CFRs were observed for 15 min followed by anintravenous bolus injection of vehicle or OM4 Fab fragment at 20 mg/kg.CFRs were then recorded for 30 min.

An un-paired t-test was used to compare the number of CFRs in saline andOM4 pre- or post-treated groups. P<0.05 was considered significant.

Pre-iniury groups. OM4 Fab fragment injected at 20 mg/kg before mechanicinjury reduced the number of CFRs from 10.5±4.1 (mean±SD) to 4.1±5.2(FIG. 15A). This reduction was statistically significant (p<0.02,t-test).

Post-injury groups. OM4 Fab fragment injected at 20 mg/kg injected afterestablishing CFR also reduced the number of CFRs from 12.2±3.8 to3.5±3.6 (FIG. 15B). This reduction was also statistically significant(p<0.003, t-test). This result demonstrates that the OM4 Fab fragmentcan inhibit thrombus formation even after initiation of the interactionbetween platelets and subendothelial collagen. Moreover, these resultssuggest that anti-GPVI antibodies can potently inhibit in vivo arterialthrombosis formation induced by endothelial injury/blood flowperturbation that are proposed triggers of thrombotic diseases inclinical situations.

EXAMPLE 10 Relationship Between the Occupancy Rate of GPVI andInhibition of Collagen-Induced Platelet Aggregation

The relationship between GPVI occupancy rate on the platelet surface andthe inhibitory effect on collagen-induced platelet aggregation bybiotinylated OM2 Fab fragment in human platelets in vitro was examined.

OM2 Fab fragment was biotinylated with Sulfo-NHS-LC-Biotin (Pierce).Biotin solution (150 μL, 10 mg/mL in distilled water) was added to OM2Fab fragment solution in PBS (5 mg, 2 mg/mL). Reaction tube wasincubated on ice for 2 hrs. To remove free biotin, the reaction mixturewas dialyzed against saline in a cold room overnight.

Blood was collected from 3 healthy donors in 1/10 volume of 3.8%trisodium citrate anticoagulant and platelet rich plasma (PRP) obtainedby centrifugation at 180×g for 15 min at room temperature. Plateletswere counted and adjusted to 3×10⁸ platelets/mL with platelet poorplasma. Measurement of aggregation was performed within 4 hrs of bloodcollection. Aggregation studies were performed in an AG10 aggregometer(Kowa, Japan). PRP was incubated with biotinylated OM2 Fab fragment (0,0.1, 0.3, 0.5, 0.7, 1, 3, 10 and 20 μg/mL) for 10 min at 37° C. beforecollagen was added and aggregation was monitored for an additional 5-10min. The collagen dose required to induce 70-90% aggregation wasdetermined for each donor prior to the evaluation of the antibody.

PRP (3×10⁸ platelets/mL, 400 μL each) was incubated with biotinylatedOM2 Fab fragment (0, 0.1, 0.3, 0.5, 0.7, 1, 3, 10 and 20 μg/mL) for 10min at room temperature. Then, washed platelets were prepared asfollows. PRP was supplemented with 1 μg/mL PGE₁, 1 mM EDTA, and EGTA andcentrifuged at 2,000×g for 10 min. After discarding plasma, the plateletpellet was suspended in platelet wash buffer (PWB: phosphate-bufferedsaline supplemented with 1 mM EDTA and EGTA, 0.1% NaN₃, 100 ng/mL PGE₁and 0.35% BSA, pH 7.4). Then, platelets were centrifuged and washedagain. The washed platelets were suspended in a small volume of PWB andplatelets were counted. Finally, the washed platelets (1×10⁸platelets/mL) were solubilized by mixing with an equal volume of 1%Triton X-100-containing phosphate-buffered saline.

Biotinylated OM2 Fab fragment was quantified by the ELISA method usingstreptavidin (Pierce, 21125) as a capture reagent and goat anti-mouseIgG antibody-HRP (American Qualex, A106PU) as a detecting reagent.

In all three donors, collagen-induced platelet aggregation was inhibitedby more than 75% by biotinylated OM2 Fab fragment at a concentration of3 μg/mL.

In the occupancy rate assay, occupancy rate was assumed to be at 100% at20 μg/mL of biotinylated OM2 Fab fragment (bound amount: 26.4±5.5ng/5×10⁷ platelets). As shown in FIG. 16, the amount of bound OM2 Fabfragment saturated at 3 μg/mL (24.3±3.5 ng/5×10⁷ platelets). Theoccupancy rate of biotinylated OM2 Fab fragment at 3 μg/mL was 92%. Thisresult suggests that an occupancy rate of more than 90% of GPVI onplatelet surface is required to exert maximal inhibitory effects oncollagen-induced platelet aggregation.

In conclusion, the GPVI specific antibodies of the invention may beuseful antithrombotic agents. As demonstrated above, the GPVI specificantibodies are potent inhibitors of platelet functions induced bycollagen.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, all of whichare hereby incorporated by reference in their entirety. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A monoclonal antibody specific for a glycoprotein VI (GPVI)polypeptide or peptide, wherein the monoclonal antibody inhibitscollagen-induced platelet aggregation at an IC₅₀ of less than 7 μg/ml,wherein the IC₅₀ is determined using a concentration of collagen thatinduces 70-90% platelet aggregation within 5 minutes of its contact withplatelets in a platelet aggregation assay, wherein the monoclonalantibody specifically binds to a GPVI polypeptide or peptide at a Kd oflower than 10⁻⁸M, and wherein the monoclonal antibody is selected fromOM1 (ATCC No. PTA-5938), OM2 (ATCC No. PTA-5939), OM3 (ATCC No.PTA-5940), and OM4 (ATCC No. PTA-5941).
 2. The monoclonal antibody ofclaim 1, wherein the monoclonal antibody is OM1.
 3. The monoclonalantibody of claim 1, wherein the monoclonal antibody is OM2.
 4. Themonoclonal antibody of claim 1, wherein the monoclonal antibody is OM3.5. The monoclonal antibody of claim 1, wherein the monoclonal antibodyis OM4.
 6. An antithrombotic composition comprising a pharmaceuticallyeffective amount of at least one monoclonal antibody of claims 1-5 and apharmaceutically acceptable excipient.
 7. A method of making anantithrombotic composition comprising admixing at least one monoclonalantibody of claims 1-5 in a pharmaceutically acceptable excipient.
 8. Akit comprising at least one dose of a pharmaceutically effective amountof at least one monoclonal antibody of claims 1-5 in a container.